PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

Leptin plays a central role in the control of energy homeostasis and appetite and, thus, has attracted attention for therapeutic approaches in spite of its limited pharmacological activity owing to the very short circulation in the body. To improve drug delivery and prolong plasma half-life, we have fused murine leptin with Pro/Ala/Ser (PAS) polypeptides of up to 600 residues, which adopt random coil conformation with expanded hydrodynamic volume in solution and, consequently, retard kidney filtration in a similar manner as polyethylene glycol (PEG). Relative to unmodified leptin, size exclusion chromatography and dynamic light scattering revealed an approximately 21-fold increase in apparent size and a much larger molecular diameter of around 18 nm for PAS(600)-leptin. High receptor-binding activity for all PASylated leptin versions was confirmed in BIAcore measurements and cell-based dual-luciferase assays. Pharmacokinetic studies in mice revealed a much extended plasma half-life after ip injection, from 26 min for the unmodified leptin to 19.6 h for the PAS(600) fusion. In vivo activity was investigated after single ip injection of equimolar doses of each leptin version. Strongly increased and prolonged hypothalamic STAT3 phosphorylation was detected for PAS(600)-leptin. Also, a reduction in daily food intake by up to 60% as well as loss in body weight of >10% lasting for >5 days was observed, whereas unmodified leptin was merely effective for 1 day. Notably, application of a PASylated superactive mouse leptin antagonist (SMLA) led to the opposite effects. Thus, PASylated leptin not only provides a promising reagent to study its physiological role in vivo but also may offer a superior drug candidate for clinical therapy.

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-fat diet induced isoform changes of the Parkinson's disease protein DJ-1.

Genetic and environmental factors mediate via different physiological and molecular processes a shifted energy balance leading to overweight and obesity. To get insights into the underlying processes involved in energy intake and weight gain, we compared hypothalamic tissue of mice kept on a high-fat or control diet for 10 days by a proteomic approach. Using two-dimensional difference gel electrophoresis in combination with LC-MS/MS, we observed significant abundance changes in 15 protein spots. One isoform of the protein DJ-1 was elevated in the high-fat diet group in three different mouse strains SWR/J, C57BL/6N, and AKR/J analyzed. Large-scale validation of DJ-1 isoforms in individual samples and tissues confirmed a shift in the pattern of DJ-1 isoforms toward more acidic isoforms in several brain and peripheral tissues after feeding a high-fat diet for 10 days. The identification of oxidation of cysteine 106 as well as 2-succinyl modification of the same residue by mass spectrometry not only explains the isoelectric shift of DJ-1 but also links our results to similar shifts of DJ-1 observed in neurodegenerative disease states under oxidative stress. We hypothesize that DJ-1 is a common physiological sensor involved in both nutrition-induced effects and neurodegenerative disease states.