Glucose enhances leptin stimulation of STAT3 phosphorylation.

<p><i>A</i>, 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. *<i>P</i><0.05. <i>B</i>, 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. <i>C</i>, 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. <i>D</i>, 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. <i>E–F</i>, PC12^LEPRb neurons (<i>E</i>) and GT1-7^LEPRb cells (<i>F</i>) 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.</p

AMPK is involved in glucose enhancement of leptin signaling.

<p><i>A</i>, 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. <i>B</i>, 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. <i>C</i>, 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.</p

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

<p><i>A–B</i>. 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. <i>C–D</i>. 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.</p

Glycolysis is required for glucose to enhance leptin signaling.

<p><i>A</i>, 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. <i>B</i>, 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. <i>C</i>. 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.</p

Glucose enhances leptin stimulation of JAK2.

<p><i>A</i>, 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. <i>B</i>, 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 <i>in vitro</i> kinase assay. The same blots were immunoblotted with αJAK2.</p

Cyclobutane Derivatives As Novel Nonpeptidic Small Molecule Agonists of Glucagon-Like Peptide-1 Receptor

A novel cyclobutane class of nonpeptidic glucagon-like peptide-1 (GLP-1) receptor agonists, exemplified by <b>3</b>, was identified using receptor binding and multiple response element/cAMP response element (MRE/CRE)-driven reporter gene assays. The structures of <b>3</b> and its three isomers were elucidated by NMR, HRESIMS, and X-ray crystallography. A series of structural modifications were also made based on the core structure of <b>3</b> with different substitution groups at the west and east ends. Among these analogues, compound <b>16</b> was found to be 4- to 5-fold more potent than <b>3</b> both in vitro and in vivo