Phosphatidylinositol 3-kinase is an upstream regulator of the phosphodiesterase 3B pathway of leptin signalling that may not involve activation of Akt in the rat hypothalamus

Abstract

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 (STAT)3, phosphatidylinositol 3-kinase (PI3K), and phosphodiesterase 3B (PDE3B)-cAMP signalling in the central nervous system 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 the anorectic and body weight reducing effects of leptin. Similarly, PI3K has been implicated to play a critical role in leptin signalling in the hypothalamus. Although, in the insulin signalling 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 signalling in the hypothalamus. To address this possibility, the effect of wortmannin, a specific PI3K inhibitor, was examined on leptin-induced PDE3B activity in the hypothalamus of male rats. Intracerebroventricular injection of leptin (4 μg) significantly increased PDE3B activity by two-fold in the hypothalamus as expected. However, previous administration of wortmannin completely reversed the stimulatory effect of leptin on PDE3B activity in the hypothalamus. To investigate whether leptin stimulates phospho (p)-Akt levels and that there might be a possible upstream regulator of PDE3B, we examined the effects of i.c.v. leptin on p-Akt levels in the hypothalamus and compared them 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 findings in that leptin failed but insulin increased the number of p-Akt positive cells in various hypothalamic nuclei. Taken together, these results implicate PI3K but not Akt as an upstream regulator of the PDE3B pathway of leptin signalling in the rat hypothalamus.

Fig. 1

Wortmannin (Wort.), a PI3K inhibitor, reverses the effect of leptin on PD3B activity in the hypothalamus. Rats fasted for 24 hours were injected ICV with DMSO or Wort. (0.01 nmol) followed 30 min later by leptin (4 μg) or aCSF (artificial cerebrospinal fluid). (A) Immunoreactive bands for PDE3B protein in the medial basal hypothalamus extracts obtained by immunoprecipitation and western blotting. PDE3B protein levels were used to normalize PDE3B activity. (B) PDE3B activity adjusted to the level of PDE3B protein in the sample and expressed as pmol/min/densitometric unit. Values are mean ± SEM for the number of animals indicated in parentheses. *P < 0.05 versus all other groups.

Fig. 2

Changes in phosphorylation of Akt at Thr³⁰⁸ or Ser⁴⁷³ and that of STAT3 in the hypothalamus at 45 min after ICV injection of leptin (4 μg), insulin (10 mU) or saline. (A) Western blot of p-Akt (Thr³⁰⁸), p-Akt (Ser⁴⁷³), Akt, p-STAT3 and STAT3. (B) Densitometric analysis of the immunoreactive bands for p-Akt (Thr³⁰⁸), p-Akt (Ser⁴⁷³) and p-STAT3. p-Akt and p-STAT3levels were first normalized to Akt and STAT3 levels, respectively, and then expressed as relative to saline control group. Values are mean ± SEM for Values are mean ± SEM for the number of animals indicated in parentheses. *P < 0.05 versus all other groups.

Fig. 3

Representative photomicrographs showing p-STAT3 positive cells in hypothalamic sections at approximately bregma -2.8(A-C) and -4.16 (D–F) from saline, leptin or insulin-treated rats that were fasted 24 hours and injected ICV with leptin (4 μg), insulin (10 mU) or saline and killed 45 min later. Scale bars, 100 μm. ARC, arcuate nucleus; VMN, ventromedial nucleus; DMN, dorsomedial nucleus; PMv, ventral premammillary nucleus; 3v, third ventricle. In this and all other figures showing immunocytochemistry, bregma are indicated in relation to the rat brain atlas (The rat brain in stereotaxic coordinates) by Paxinos and Watson (Ref. 26).

Fig. 4

Representative photomicrographs showing p-Akt (Ser⁴⁷³) positive cells in the hypothalamic sections at approximately bregma −2.0 (A–C) and −2.8 (D–F) from saline, leptin or insulin-treated rats that were fasted 24 hours and injected ICV with leptin (4 μg), insulin (10 mU) or saline and killed 45 min later. Scale bars, 100 μm. PVN, paraventricular nucleus; ARC, arcuate nucleus; VMN, ventromedial nucleus; DMN, dorsomedial nucleus; 3v, third ventricle.

Fig. 5

Representative photomicrographs of p-Akt (Ser⁴⁷³) ICC of hypothalamic sections at approximately bregma −4.16 (A–C) and −3.8 (D–F) from saline, leptin or insulin-treated rats that were fasted 24 hours and injected ICV with leptin (4 μg), insulin (10 mU) or saline and killed 45 min later. Scale bars, 100 μm. ARC, arcuate nucleus; PMv, ventral premammillary nucleus; VTM, ventral tuberomammillary nucleus; 3v, third ventricle.

Fig. 6

Changes in the number of p-Akt (Ser⁴⁷³) positive cells in various hypothalamic sites of saline, leptin or insulin-treated rats. One of six series from each animal was analyzed. ARC, arcuate nucleus; VMN, ventromedial nucleus; DMN, dorsomedial nucleus; LH, lateral hypothalamus; PVN, paraventricular nucleus; PMv, ventral premammillary nucleus; VTM, ventral tuberomammillary nucleus. R, M and C for respective nuclei represent rostral, middle and caudal part, respectively. Values represent the mean ± SEM for 3 animals per group.*P < 0.05 vs. saline group.

Fig. 7

Changes in phosphorylation of Akt at Thr³⁰⁸ or Ser⁴⁷³ and that of STAT3 in the hypothalamus at 10 min after ICV injection of leptin (4 μg), insulin (10 mU) or saline. (A) Western blot of p-Akt (Thr³⁰⁸), p-Akt (Ser⁴⁷³), and Akt, p-STAT3 and STAT3. (B) Densitometric analysis of the immunoreactive bands for p-Akt (Thr³⁰⁸). (C) Densitometric analysis of the immunoreactive bands for p-Akt (Ser⁴⁷³). (D) Western blot of p-STAT3 and STAT3. (E) Densitometric analysis of the immunoreactive bands for p-STAT3. p-Akt and p-STAT3 levels were first normalized to Akt and STAT3 levels, respectively, and then expressed as relative to saline control group. Values are mean ± SEM for Values are mean ± SEM for the number of animals indicated in parentheses. *P < 0.05 versus all other groups.

Fig. 8

Representative photomicrographs of p-Akt (Ser⁴⁷³) positive cells (A–C) and p-STAT3 positive cells (D–F) in the hypothalamic sections at approximately bregma −2.8 from saline, leptin or insulin-treated rats. Rats fasted for 24 hours were injected ICV with leptin (4 μg), insulin (10 mU) or saline and killed 10 min later. Scale bars, 100 μm. ARC, arcuate nucleus; VMN, ventromedial nucleus; DMN, dorsomedial nucleus; 3v, third ventricle.

Fig. 9

Representative photomicrographs of p-Akt (Ser⁴⁷³) positive cells (A–C, G–I) and p-STAT3 positive cells (D–F) in the hypothalamic sections at approximately bregma −3.8 (A–C), and bregma −4.16 (D–I) from saline, leptin or insulin-treated rats. Rats fasted for 24 hours were injected ICV with leptin (4 μg), insulin (10 mU) or saline and killed 10 min later. Scale bars, 100 μm. ARC, arcuate nucleus; PMv, ventral premammillary nucleus; VTM, ventral tuberomammillary nucleus; 3v, third ventricle.