Glucose enhances leptin signaling through modulation of AMPK activity

Abstract

Leptin exerts its action by binding to and activating the long form of leptin receptors (LEPRb). LEPRb activates JAK2 that subsequently phosphorylates and activates STAT3. The JAK2/STAT3 pathway is required for leptin control of energy balance and body weight. Defects in leptin signaling lead to leptin resistance, a primary risk factor for obesity. Body weight is also regulated by nutrients, including glucose. Defects in glucose sensing also contribute to obesity. Here we report crosstalk between leptin and glucose. Glucose starvation blocked the ability of leptin to stimulate tyrosyl phosphorylation and activation of JAK2 and STAT3 in a variety of cell types. Glucose dose-dependently enhanced leptin signaling. In contrast, glucose did not enhance growth hormone-stimulated phosphorylation of JAK2 and STAT5. Glucose starvation or 2-deoxyglucose-induced inhibition of glycolysis activated AMPK and inhibited leptin signaling; pharmacological inhibition of AMPK restored the ability of leptin to stimulate STAT3 phosphorylation. Conversely, pharmacological activation of AMPK was sufficient to inhibit leptin signaling and to block the ability of glucose to enhance leptin signaling. These results suggest that glucose and/or its metabolites play a permissive role in leptin signaling, and that glucose enhances leptin sensitivity at least in part by attenuating the ability of AMPK to inhibit leptin signaling.

Figure 1. Glucose enhances leptin stimulation of STAT3 phosphorylation.

A, γ2A^LEPRb/JAK2 cells were grown overnight in serum-free medium supplemented with 5 or 25 mM glucose and treated with 100 ng/ml leptin for 10 min. Cell extracts were immunoblotted with anti-phospho-STAT3 (pTyr⁷⁰⁵) (αpSTAT3) or αSTAT3 antibodies. The amounts of phospho-STAT3 and total STAT3 were quantified using densitometry, and STAT3 phosphorylation was normalized to the total amount of STAT3. *P<0.05. B, γ2A^LEPRb/JAK2 cells were deprived of serum in the presence of 5 mM glucose overnight. Cells were pretreated with 25 mM glucose for 0, 10, 30, 60, 120 or 240 min, and then treated with 100 ng/ml leptin for 10 min. Cell extracts were immunoblotted with αpSTAT3 or αSTAT3. C, γ2A^LEPRb/JAK2 cells were deprived of serum overnight in the presence of 0, 5, 10, 15 or 25 mM glucose, and stimulated with 100 ng/ml leptin for 10 min. Cell extracts were immunoblotted with αpSTAT3 or αSTAT3. D, γ2A^LEPRb/JAK2 cells were deprived of serum in the presence of 5 or 25 mM glucose overnight, and then stimulated with leptin for 10 min at various concentrations. Cell extracts were immunoblotted with αpSTAT3 or αSTAT3, respectively. E–F, PC12^LEPRb neurons (E) and GT1-7^LEPRb cells (F) were deprived of serum overnight in 5 or 25 mM glucose and then treated with 100 ng/ml leptin for 10 min. Cell extracts were immunoblotted with αpSTAT3 or αSTAT3.

Figure 2. Glucose enhances leptin stimulation of JAK2.

A, γ2A^LEPRb/JAK2 cells were deprived of serum in the presence of 5 or 25 mM glucose overnight and then treated with 100 ng/ml leptin for 10 min. Cell extracts were immunoblotted with anti-phospho-JAK2 (pTyr^1007/1008) (αpJAK2), αJAK2, αpSTAT3, or αSTAT3 as designated. B, γ2A^LEPRb/JAK2 cells were treated with 5 or 25 mM glucose overnight and then with 100 ng/ml leptin for 10 min. JAK2 in cell extracts was immunoprecipitated with αJAK2 and subjected to an in vitro kinase assay. The same blots were immunoblotted with αJAK2.

Figure 3. Glucose does not enhance GH-stimulated phosphorylation of JAK2 and STAT5.

A, γ2A^GHR/JAK2 cells were deprived of serum in the presence of 5 or 25 mM glucose overnight and then treated with GH for 15 min. Cell extracts were immunoblotted with αpSTAT5, αSTAT5, αpJAK2 or αJAK2, respectively. B, 3T3-F442A cells were treated with 5 or 25 mM glucose overnight and then with 50 ng/ml GH for 15 min. Cell extracts were immunoblotted with the indicated antibodies.

Figure 4. Glycolysis is required for glucose to enhance leptin signaling.

A, γ2A^LEPRb/JAK2 cells were grown overnight in serum-free medium supplemented with 25 mM D-glucose, 5 mM D-glucose plus 20 mM L-glucose, or 5 mM D-glucose plus 20 mM sorbitol. Cells were stimulated with 100 ng/ml leptin for 10 min, and cell extracts were immunoblotted with αpSTAT3 or αSTAT3. B, γ2A^LEPRb/JAK2 cells were grown overnight in serum-free medium supplemented with 25 mM D-glucose, pretreated with 25 mM 2-DG for 3 h, and then treated with 100 ng/ml leptin for 10 min. Cell extracts were immunoblotted with αpSTAT3 or αSTAT3. C. γ2A^LEPRb/JAK2 cells were grown overnight (∼15 h) in serum-free medium containing 5 mM D-glucose plus additional 20 mM lactate, pyruvate, or D-glucose, and stimulated with 100 ng/ml leptin for 10 min. Cell extracts were immunoblotted with αpSTAT3 or αSTAT3.

Figure 5. Oxidative stress, the mTOR pathway, and the p38 MAPK pathway do not mediate glucose enhancement of leptin signaling.

A–B. γ2A^LEPRb/JAK2 cells were grown overnight in serum-free medium supplemented with 5 or 25 mM D-glucose, 10 mM NAc (A) or 200 µM H₂O₂ (B). Cells were stimulated with 100 ng/ml leptin for 10 min, and cell extracts were immunoblotted with αpSTAT3 or αSTAT3. C–D. γ2A^LEPRb/JAK2 cells were incubated in 5 or 25 mM glucose overnight, pretreated with 50 nM rapamycin for 1 h (C) or 2 mM L-leucine for 2 h (D), and then stimulated with 100 ng/ml leptin for 10 min. Cell extracts were immunoblotted with αpSTAT3 or αSTAT3.

Figure 6. AMPK is involved in glucose enhancement of leptin signaling.

A, γ2A^LEPRb/JAK2 cells were deprived of serum overnight (in 25 mM glucose). Cells were treated with 25 mM 2-DG or 2 mM AICAR for 3 h, and then with 100 ng/ml leptin for additional 10 min. Cell extracts were immunoblotted with the indicated antibodies. B, PC12^LEPRb neurons were deprived of serum overnight in the presence of 25 mM glucose, and treated with 25 mM 2-DG or 2 mM AICAR for 1 h and then with 100 ng/ml leptin for additional 10 min. Cell extracts were immunoblotted with the indicated antibodies. C, PC12^LEPRb neurons were incubated overnight (∼15 h) in the presence or absence of 40 µM compound C. Some cells were pretreated with 2-DG (25 mM) for 1 h as indicated. Cells were stimulated with 100 ng/ml leptin for 10 min, and cell extracts were immunoblotted with the indicated antibodies.