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

The opposite effect of glucoprivation on sEPSC distributions in EPSC(+) and EPSC(-) neurons.

<p>Spontaneous EPSCs were recorded in POMC neurons at baseline 5 mM glucose or 0.1 mM glucose (perfused with 0.1 mM glucose for 16 min) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105080#pone-0105080-g002" target="_blank">Figure 2</a>. (A) The inter-event distribution curves of sEPSCs. (B) The amplitude distribution curves of sEPSCs. EPSC(+): n = 9, EPSC(−): n = 6.</p

Glucose-excited POMC neurons in the ARC.

<p>(A) Hypothalamic sections containing the ARC were prepared from POMC DsRed mice (7 weeks), and POMC neurons were visualized using a fluorescent microscope. (B) Action potentials were recorded in the whole-cell current-clamp mode (injecting 10 pA depolarizing currents) in POMC neurons perfused sequentially with aCSF containing 5 mM glucose (baseline), 0.1 mM glucose, and 5 mM glucose (washout).</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

The bi-phasic effects of glucoprivation on sEPSC distributions in EPSC(+/−) neurons.

<p>(A) The inter-event interval distribution curves of sEPSCs in EPSC(+/−) neurons at baseline, 0.1 mM glucose for 15 min, or 0.1 mM glucose for 30 min. (B) The amplitude distribution curves of sEPSCs in EPSC(+/−) neurons at baseline, 0.1 mM glucose for 15 min, or 0.1 mM glucose for 30 min. EPSC(+/−): n = 5.</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 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

A model of glucose-regulated excitatory synaptic transmission in POMC neurons.

<p>(A) A EPSC(+) neuron was innervated directly by a glutamatergic, glucose-excited (GE) neuron (pink), and extracellular glucose stimulates the GE neuron to increase glutamatergic inputs onto the EPSC(+) neuron. Additionally, the EPSC(+) neuron may also be innervated indirectly by a GABAergic, glucose-inhibited (GI) neuron (blue) via a glutamatergic neuron (pink), and extracellular glucose causes disinhibition of the EPSC(+) neuron through this circuit. (B) A EPSC(−) neuron was innervated by a glutamatergic GI neuron, and glucose inhibits glutamatergic transmission in the EPSC(−) neuron. Additionally, the EPSC(−) neuron may also be innervated indirectly by a GABAergic GE neuron via a glutamatergic neuron. Glucose inhibits this glutamatergic neuron by exciting the inhibitory GE neuron, thus decreasing the frequency of sEPSCs in the EPSC(−) neuron. (C) A EPSC(+/−) neuron is regulated by EPSC(+)-like connections during the first phase of glucoprivation (left) and by EPSC(−)-like connections during the second phase (right).</p

Liver-specific deletion of <i>SH2B1</i> does not augment MCD-induced liver injury.

<p>Male mice (7 weeks of age) were fed a MCD. (A) Growth curves. <i>f/f</i>: n = 5; HKO: n = 7. (B) Overnight fasting blood glucose. <i>f/f</i>: n = 5; HKO: n = 5. (C) ITT (MCD for 7 weeks). <i>f/f</i>: n = 5; HKO: n = 5. (D) GTT (MCD for 6 weeks). <i>f/f</i>: n = 5; HKO: n = 5. (E) Plasma ALT activity. <i>f/f</i>: n = 5; HKO: n = 5. (F). Plasma ALP activity. <i>f/f</i>: n = 5; HKO: n = 5. (G) Plasma bilirubin levels (MCD for 8 weeks). <i>f/f</i>: n = 5; HKO: n = 5. (H) Liver TAG levels (MCD for 8 weeks). <i>f/f</i>: n = 5; HKO: n = 5. Data are presented as means ± SEM. <i>P</i><0.05.</p