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

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

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

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

Enu Mutagenesis Identifies a Novel Platelet Phenotype in a Loss-Of-Function Jak2 Allele

<div><p>Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (<i>Jak2</i>^<i>K915X</i>), resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (<i>Jak2</i>^<i>+/-</i>). Timed pregnancy experiments revealed that <i>Jak2</i>^{<i>K915X/K915X</i>} and <i>Jak2</i>^<i>-/-</i> displayed embryonic lethality; however, <i>Jak2</i>^{<i>K915X/K915X</i>} embryos were viable an additional two days compared to <i>Jak2</i>^<i>-/-</i> embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.</p> </div

Strain 7254 is defined by a K915X mutation and generates a non-functional truncated Jak2 protein.

<p>(A) The Jak2 domain structure is indicated. The DNA sequence from <i>7254</i> splenocytes and corresponding protein sequence are also shown. (B) Splenocytes were harvested from phenylhydrazine primed <i>Jak2</i>^<i>K915X</i> and <i>Jak2</i>^<i>+/-</i> mice and their wild type littermates. HA-tagged Jak2 and Jak2^K915X were also expressed in 293T cells. A Western blot was performed with a peptide-specific JAK2 antibody. (C) 293T cells were transfected with cDNAs encoding HA-Jak2 or HA-Jak2 K915X. Western blotting was performed with phosphorylation-specific antibodies that recognize pSer-523, pTyr-570 and pTyr-1007/1008 in Jak2. The membranes were stripped and reprobed with an anti-HA antibody. (D) HA-tagged versions of Jak2 and Jak2^K915X with or without the JAK2 3’ UTR were expressed in 293T cells. Western blots were performed with 4G10 anti-phosphotyrosine and HA antibodies. Immunoblotting with anti β-tubulin was performed to demonstrate equal loading.</p

The K915X mutation in Jak2 enhances viability of mouse embryos.

<p>Embryos from timed matings were sacrificed at E12.5-E14.5. Representative embryos from E12.5 are shown. A summary of the results is provided in tabular format.</p

<i>Jak2</i>^{<i>K915X/+</i>} mice have elevated megakaryocytes and platelets.

<p>(A) Bone marrow sections were prepared from 12 week <i>Jak2</i>^<i>+/+</i> and <i>Jak2</i>^{<i>K915X/+</i>} mice and stained with H and E. Representative sections are illustrated at 20x magnification. Megakaryocytes are indicated by an asterisk. (B) Platelets from male and female wild type, <i>Jak2</i>^<i>+/-</i> and <i>Jak2</i>^<i>K915X</i> mice at 8 wk of age were monitored. (C) Red blood cells were evaluated from male and female mice at 8 wk of age from WT, <i>Jak2</i>^<i>+/-</i> and <i>Jak2</i>^{<i>K915X/+</i>} mice. <i>Jak2</i>^<i>+/+</i> or <i>JAK2</i>^{<i>Control</i>} mice were littermate controls of <i>Jak2</i>^<i>+/-</i> or <i>Jak2</i>^{<i>K915X/+</i>} breedings, respectively. Statistically significant differences between groups are denoted as *, p< 0.05 and **, p<0.0001. Each group has n=20-30.</p