Phosphatidylinositol 3-kinase (PI3K) pathway of leptin signaling plays an important role in transducing leptin action in the hypothalamus. Obesity is usually associated with resistance to the effect of leptin on food intake and energy homeostasis. Although central leptin resistance is thought to be involved in the development of diet-induced obesity (DIO), the mechanism behind this phenomenon is not clearly understood. To determine whether DIO impairs the effect of leptin on hypothalamic PI3K signaling, we fed 4-wk-old FVB/N mice a high-fat diet (HFD) or low-fat diet (LFD) for 19 wk. HFD-fed mice developed DIO in association with hyperleptinemia, hyperinsulinemia, and impaired glucose and insulin tolerance. Leptin (ip) significantly increased hypothalamic PI3K activity and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) levels in LFD-fed mice but not in DIO mice. Immunocytochemical study confirmed impaired p-STAT3 activation in various hypothalamic areas, including the arcuate nucleus. We next tested whether both PI3K and STAT3 pathways of leptin signaling were impaired during the early period of DIO. Leptin failed to increase PI3K activity in DIO mice that were on a HFD for 4 wk. However, leptin-induced p-STAT3 activation in the hypothalamus measured by Western blotting and immunocytochemistry remained comparable between LFD- and HFD-fed mice. These results suggest that the PI3K pathway but not the STAT3 pathway of leptin signaling is impaired during the development of DIO in FVB/N mice. Thus, a defective PI3K pathway of leptin signaling in the hypothalamus may be one of the mechanisms of central leptin resistance and DIO.

Leptin signaling in the hypothalamus is obligatory for normal food intake and body weight homeostasis. It is now well established 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 bodyweight reducing effects of leptin. Recently, we have demonstrated that cilostamide reversed the leptin-induced increase in proopiomelanocortin (POMC) gene expression in the hypothalamus. Because POMC and neuropeptide Y (NPY) neurons are thought to be the major targets of leptin signaling in the hypothalamus, to establish the physiological role of the PDE3B pathway it is important to demonstrate if PDE3B is expressed in these neurons. To this end we examined co-localization of PDE3B with POMC and NPY neurons using immunocytochemistry in POMC-GFP and NPY-GFP mice, respectively. Results showed that PDE3B was highly localized throughout the various hypothalamic sites including the arcuate nucleus (ARC), ventromedial nucleus, dorsomedial nucleus, ventral premammillary nucleus, paraventricular nucleus, and lateral hypothalamus. Importantly, almost all NPY (91.7%) and POMC (97.7%) neurons co-expressed PDE3B. These results suggest a direct role of the PDE3B pathway in mediating leptin signaling in the POMC and NPY neurons –a potential mechanism of leptin signaling in the hypothalamus.

Leptin, the product of the obese gene, regulates energy homeostasis by acting primarily at the level of the hypothalamus. Leptin action through its receptor involves various pathways including the signal transducer and activator of transcription (STAT3), phosphatidylinositol 3-kinase (PI3K), and phosphodiesterase 3B (PDE3B)-cAMP signaling in the CNS and peripheral tissues. In the hypothalamus, leptin stimulates STAT3 activation, and induces PI3K and PDE3B activities, among others. We have previously demonstrated that PDE3B activation in the hypothalamus is critical for transducing anorectic and body weight reducing effects of leptin. Similarly, PI3K has been implicated toplay a critical role in leptin signaling in the hypothalamus. Whereas in insulin signaling pathway, PI3K is known to be an upstream regulator of PDE3B in non-neuronal tissues, it is still unknown whether this is also the case for leptin signaling in the hypothalamus. To address this possibility, the effect of wortmannin, a specific PI3K inhibitor, was examined on the leptin-induced PDE3B activity in the hypothalamus of male rats. Intracerebroventricular (icv) injection of leptin (4 μg) significantly increased PDE3B activity by 2-fold in the hypothalamus as expected. However, prior administration of wortmannin completely reversed the stimulatory effect of leptin on PDE3B activity in the hypothalamus. To demonstrate whether leptin stimulates p-Akt levels and there by a possible upstream regulator of PDE3B, we examined the effects of icv leptin on p-Akt levels in the hypothalamus and compared that with the known stimulatory effect of insulin on p-Akt. We observed that insulin increased p-Akt levels but leptin failed to do so although it increased p-STAT3 levels in the rat hypothalamus. Immunocytochemistry confirmed the biochemical finding in that leptin failed but insulin increased the number of p-Akt positive cells in various hypothalamic nuclei. Altogether these results implicate PI3K but not Akt as an upstream regulator of the PDE3B pathway of leptin signaling in the rat hypothalamus.

The phosphodiesterase-3B (PDE3B)-cAMP pathway plays an important role in transducing the action of leptin in the hypothalamus. Obesity is usually associated with hyperleptinaemia and resistance to anorectic and body weight-reducing effects of leptin. To determine whether the hypothalamic PDE3B-cAMP pathway of leptin signalling is impaired during the development of diet-induced obesity (DIO), we fed male FVB/N mice a high-fat diet (HFD: 58% kcal as fat) or low-fat diet (LFD: 6% kcal as fat) for 4 weeks. HFD fed mice developed DIO in association with hyperphagia, hyperleptinaemia and hyperinsulinaemia. Leptin (i.p.) significantly increased hypothalamic PDE3B activity and phosphorylated (p)-Akt levels in LFD-fed but not in HFD-fed mice. However, basal p-Akt levels in hypothalamus were increased in DIO mice. Additionally, amongst six-microdissected brain nuclei examined, leptin selectively decreased cAMP levels in the arcuate nucleus (ARC) of LFD-fed mice but failed to do so in HFD-fed mice. We next tested whether both the PBE3B and Akt pathways of leptin signalling remained impaired in DIO mice on the HFD for 12 weeks (long-term). DIO mice were hyperinsulinaemic and hyperleptinaemic in association with impaired glucose and insulin tolerance. Although, in LFD-fed mice, leptin significantly increased PDE3B activity and p-Akt levels in the hypothalamus, it failed to do so in HFD-fed mice. Also, basal p-Akt levels in the hypothalamus were increased in DIO mice and leptin had no further effect. Similarly, immunocytochemistry showed that leptin increased the number of p-Akt-positive cells in the ARC of LFD-fed but not in HFD-fed mice, and there was an increased basal number of p-Akt positive cells in the ARC of DIO mice. These results suggest that the PDE3B-cAMP- and Akt-pathways of leptin signalling in the hypothalamus are impaired during the development of DIO. Thus, a defect in the regulation by leptin of the hypothalamic PDE3B-cAMP pathway and Akt signalling may be one of the mechanisms of central leptin resistance and the development of DIO.