Intracellular leptin-signaling pathways in hypothalamic neurons: the emerging role of phosphatidylinositol-3 kinase-phosphodiesterase-3B-cAMP pathway

Abstract

Leptin is secreted primarily by fat cells and acts centrally, particularly in the hypothalamus, to reduce food intake and body weight. Besides the classical JAK2 (Janus kinase-2)-STAT3 (signal transducer and activator of transcription-3) pathway, several non-STAT3 pathways play an important role in mediating leptin signaling in the hypothalamus. We have demonstrated that leptin action in the hypothalamus is mediated by an insulin-like signaling pathway involving stimulation of PI3K (phosphatidylinositol-3 kinase) and PDE3B (phosphodiesterase-3B), and reduction in cAMP levels, and that a PI3K-PDE3B-cAMP pathway interacting with the JAK2-STAT3 pathway constitutes a critical component of leptin signaling in the hypothalamus. It appears that defective regulation of multiple signaling pathways in the hypothalamus causes central leptin resistance, a major cause of obesity. In this regard, we have shown that leptin resistance in hypothalamic neurons following chronic central infusion of this hormone is associated with a defect in the PI3K-PDE3B-cAMP, and not due to compromised signaling in the JAK2-STAT3 pathway. Similarly, the PI3K, but not the STAT3, pathway is impaired in the hypothalamus during the development of diet-induced obesity. Additionally, our recent work suggests that suppressor of cytokine signaling-3 negatively regulates the PI3K pathway of leptin signaling in the hypothalamus, a mechanism expected to play a significant role in diet-induced obesity. Together, the PI3K-PDE3B-cAMP pathway appears to emerge as a major mechanism of leptin signaling in the hypothalamus in regulating energy balance.

Fig. 1

Schematic presentation of leptin action on hypothalamic signals governing feeding. For details, see the text. NPY = Neuropeptide Y; MCH = melanin-concentrating hormone; POMC = proopiomelanocortin; NT = neurotensin [modified from [4]].

Fig. 2

Cilostamide, a specific PDE3 inhibitor, reverses the effect of leptin on STAT3 activation (DNA binding activity) in the hypothalamus. Fasted (24 h) rats were injected intracerebroventricularly with DMSO (vehicle for cilostamide) or cilostamide (10 μg) followed 30 min later by leptin (4 μg) or artificial cerebrospinal fluid (aCSF, vehicle for leptin). Top: DNA binding activity of STAT3 in the medial basal hypothalamic (MBH) extracts as determined by an electrophoretic mobility shift assay using a ³²P-labeled M67-SIE oligonucleotide probe. Bottom: results obtained by phosphor imaging and expressed as relative (%) to vehicle. Data are means ± SEM for the number of animals in parentheses. ^* p < 0.05 as compared to all other groups. The reversal of STAT3 activation by PDE3B inhibition implies a crosstalk between the JAK2-STAT3 and PDE3B-cAMP pathways in transducing leptin action [modified from [21]].

Fig. 3

Cilostamide reverses leptin-induced proopiomelanocortin (POMC) and neurotensin (NT) gene expression as determined by ribonuclease protection assay in the hypothalamus. Rats were injected with cilostamide (10 μg/1 μl) in dimethyl sulfoxide (DMSO) or DMSO alone, and recombinant murine leptin (4 μg/ 2 μl) in artificial cerebrospinal fluid (aCSF) or aCSF alone, at 17:00–18:00 h, and food was withdrawn. One hour later, another injection of cilostamide or DMSO was given to the rats. After 24 h, a similar injection protocol was used and rats were sacrificed 15 h after the last injection. Values represent the means ± SEM. Control (aCSF + DMSO): n = 4, leptin: n = 5, cilostamide: n = 5, and leptin + cilostamide: n = 7. ^* p < 0.05 and ^** p < 0.01 vs. all other groups [adapted from [44]].

Fig. 4

Schematic of leptin intracellular signaling through the STAT3 and PI3K-PDE3B-cAMP pathways in the hypothalamus. Leptin binding to its receptor (Ob-Rb) induces activation of JAK2, receptor dimerization, and JAK2-mediated phosphorylation of intracellular part of Ob-Rb, followed by phosphorylation and activation of STAT3. Activated STAT3 dimerizes and translocates to the nucleus and transactivates target genes including SOCS3, NPY and POMC. Our evidence suggests that leptin also activates PI3K and PDE3B, and reduces cAMP levels. Decrease in cAMP levels appears to be important for STAT3 activation by leptin, because PDE3 inhibition reverses this effect of leptin in the hypothalamus. Also, leptin-induced PDE3B activation-dependent decrease in cAMP levels may directly modify (increase or decrease) expression of POMC, NT, NPY and other genes – a hypothesis needs to be experimentally tested. SOCS3 acts as a negative regulator of both the STAT3 and PI3K pathways of leptin signaling in the hypothalamus, a mechanism that plays a significant role in the development of central leptin resistance and DIO. Other pathways such as SHP2, MAPK/ERK, AMPK, and mTOR pathways and PTP1B that are known to play a role in regulating leptin action and leptin resistance in the hypothalamus have been left out to simplify the figure [modified from [4]].

Fig. 5

Leptin failed to increase IRS-1 associated PI3K activity but it increased p-STAT3 in the hypothalamus at 4 weeks on a high-fat diet (HFD) in FVB/N mice. Male mice (∼4 weeks old) were fed with either a low-fat diet (LFD) or a HFD. Four weeks later, overnight-fasted animals were injected with leptin (5 mg/kg, i.p.) or saline. PI3K and p-STAT3 were assessed in the hypothalamus after 30 min of injection. a Representative phosphor images obtained from the TLC plate showing PI3P production, an indicator of PI3K activity, as shown. Also shown are p-STAT3 and STAT3 Western blots obtained from the hypothalamic extract used in the PI3K assay. b Results obtained by phosphor imaging showing the changes in PI3K activity. The values are expressed as relative to the corresponding saline (control) group. ^** p < 0.01 (n = 6 per group). c Densitometric analysis of the immunoreactive bands for p-STAT3. The values were first calculated as the ratio of STAT3 and then expressed as relative to the corresponding saline (control) group. ^* p < 0.05 vs. saline group (n = 5–6 per group). d Representative micrographs of p-STAT3 immunocytochemistry of hypothalamic sections showing leptin-induced increase in p-STAT3-positive cells in both LFD- and HFD-fed mice. Scale bars = 100 μm. ME = Median eminence; ARC = arcuate nucleus; 3v = third ventricle [adapted from [48]].