leptin and cilostamide

Insulin action in the hypothalamus plays a critical role in the regulation of energy homeostasis, yet the intracellular signaling mechanisms mediating insulin action are incompletely understood. Although phosphodiesterase 3B (PDE3B) mediates insulin action in the adipose tissue and it is highly expressed in the hypothalamic areas implicated in energy homeostasis, its role, if any, in mediating insulin action in the hypothalamus is unknown. We tested the hypothesis that insulin action in the hypothalamus is mediated by PDE3B.. Using enzymatic assay, we examined the effects of peripheral or central administration of insulin on hypothalamic PDE3B activity in adult mice. Western blotting and immunohistochemistry also examined p-Akt and p-STAT3 levels in the hypothalamus. Effects of leptin on these parameters were also compared. We injected cilostamide, a PDE3 inhibitor, prior to central injection of insulin and examined the 12- to 24-hour food intake and 24-hour body weight. Finally, we examined the effect of cilostamide on insulin-induced proopiomelanocortin (Pomc), neurotensin (Nt), neuropeptide Y (Npy) and agouti-related peptide (Agrp) gene expression in the hypothalamus by qPCR.. Peripheral or central injection of insulin significantly increased PDE3B activity in the hypothalamus in association with increased p-Akt levels but without any change in p-STAT3 levels. However, leptin-induced increase in PDE3B activity was associated with an increase in both p-Akt and p-STAT3 levels in the hypothalamus. Prior administration of cilostamide reversed the anorectic and body weight-reducing effects as well as stimulatory effect of insulin on hypothalamic Pomc mRNA levels. Insulin did not alter Nt, Npy and Agrp mRNA levels.. Insulin induction of hypothalamic PDE3B activity and the reversal of the anorectic and body weight-reducing effects and stimulatory effect of insulin on hypothalamic Pomc gene expression by cilostamide suggest that activation of PDE3B is a novel mechanism of insulin signaling in the hypothalamus.

Topics: Animals; Body Weight; Cyclic Nucleotide Phosphodiesterases, Type 3; Eating; Gene Expression; Hypothalamus; Insulin; Leptin; Male; Mice; Mice, Inbred C57BL; Phosphodiesterase Inhibitors; Phosphorylation; Pro-Opiomelanocortin; Proto-Oncogene Proteins c-akt; Quinolones; Signal Transduction; STAT3 Transcription Factor; Weight Loss

Leptin signaling in the hypothalamus is required for normal food intake and body weight homeostasis. Recent evidence suggests that besides the signal transducer and activator of transcription-3 (STAT3) pathway, several non-STAT3 pathways mediate leptin signaling in the hypothalamus. We have previously demonstrated that leptin stimulates phosphodiesterase-3B (PDE3B) activity in the hypothalamus, and PDE3 inhibitor cilostamide reverses anorectic and body weight reducing effects of leptin. To establish the physiological role of PDE3B signaling in the hypothalamus, we examined if leptin signaling through the PDE3B pathway is responsible for the activation of proopiomelanocortin (POMC) and neurotensin (NT) neurons, which are known to play a critical role in energy homeostasis. To this end, we assessed the effect of cilostamide on leptin-induced POMC and NT gene expression in the rat hypothalamus. Results showed that while central injection of leptin significantly increased both POMC and NT mRNA levels in the medial basal hypothalamus, cilostamide completely reversed this effect of leptin suggesting a PDE3B-activation dependent induction of POMC and NT gene expression by leptin. This result further suggests that the PDE3B pathway plays an important role in mediating leptin action in the hypothalamus.

Topics: Animals; Cyclic Nucleotide Phosphodiesterases, Type 3; Gene Expression; Hypothalamus; Leptin; Male; Models, Neurological; Neurons; Neurotensin; Phosphodiesterase 3 Inhibitors; Phosphodiesterase Inhibitors; Pro-Opiomelanocortin; Quinolones; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction

Obesity is associated with the development of leptin resistance. However, the effects of leptin resistance on leptin-regulated metabolic processes and the biochemical defects that cause leptin resistance are poorly understood. We have addressed in rats the effect of dietinduced obesity (DIO), a situation of elevated tissue lipid levels, on the well described lipid-lowering effect of leptin in liver, an action that is proposed to be important for the prevention of tissue lipotoxicity and insulin resistance. In addition, we have addressed the role of phosphatidylinositol 3-kinase (PI 3-kinase) in mediating the acute effects of leptin on hepatic lipid levels in lean and DIO animals. A 90-min leptin ( approximately 10 ng/ml) perfusion of isolated livers from lean animals decreased triglyceride levels by 42 +/- 5% (p = 0.006). However, leptin concentrations ranging from approximately 10 to approximately 90 ng/ml had no effect on triglyceride levels in livers from DIO animals. The acute lipid-lowering effect of leptin on livers from lean animals was mediated by a PI 3-kinase-dependent mechanism, because wortmannin and LY294002, the PI 3-kinase inhibitors, blocked the effects of leptin on hepatic triglyceride levels and leptin increased liver PI 3-kinase activity by 183 +/- 6% (p = 0.003) and insulin receptor substrate 1 tyrosine phosphorylation by 185 +/- 30% (p = 0.02) in the absence of PI 3-kinase inhibitors. Contrary to the effects of leptin in lean livers, leptin did not activate PI 3-kinase in livers from DIO rats. These data present evidence for a role for 1). leptin resistance in contributing to the excessive accumulation of tissue lipid in obesity, 2). PI 3-kinase in mediating the acute lipid-lowering effects of leptin in liver, and 3). defective leptin activation of PI 3-kinase as a novel mechanism of leptin resistance.

Topics: Androstadienes; Animals; Chromones; Diet; Dose-Response Relationship, Drug; Enzyme Inhibitors; Insulin Resistance; Leptin; Lipid Metabolism; Liver; Male; Morpholines; Obesity; Oxygen Consumption; Perfusion; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Quinolones; Rats; Rats, Wistar; Serine; Time Factors; Triglycerides; Wortmannin

Using male Sprague-Dawley rats implanted with third intracerebroventricular (ICV) cannulae, we found that cilostamide, a phosphodiesterase 3 (PDE3) inhibitor, (i) reversed the established effects of leptin on food intake and body weight, (ii) blocked, at the hypothalamic level, the leptin-induced tyrosine phosphorylation of signal transducer and activator of transcription 3 (Stat3) and (iii) blocked the DNA binding of p-Stat3. Additionally, ICV administration of leptin increased hypothalamic phosphatidylinositol 3-kinase (PI3K) and PDE3B activities and decreased cyclic AMP (cAMP) concentration. These results indicate that a PI3K-PDE3B-cAMP pathway interacting with the Janus kinase 2 (Jak2)-Stat3 pathway constitutes a critical component of leptin signaling in the hypothalamus.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Body Weight; Cyclic AMP; Cyclic Nucleotide Phosphodiesterases, Type 3; DNA; DNA-Binding Proteins; Electrodes, Implanted; Enzyme Activation; Enzyme Inhibitors; Feeding Behavior; Hypothalamus; Injections, Intraventricular; Janus Kinase 2; Leptin; Male; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Binding; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Quinolones; Rats; Rats, Sprague-Dawley; Signal Transduction; STAT3 Transcription Factor; Trans-Activators