Altered leptin signaling in POUND mice alters IGF-1 signaling in skeletal muscle.

<p>A. ELISA assays show that muscle-derived IGF-1 is significantly decreased in the hindlimb muscles (extensor digitorum longus) from leptin receptor-deficient POUND mice (left graph), whereas protein levels of myostatin in hindlimb muscle are significantly elevated in POUND mice (right graph). B. Integrated pathway analysis from mRNA array comparing gene expression in tibialis anterior muscles of POUND mice with that of normal mice. The vertical axis represents the probability that a particular gene is associated with a specific canonical pathway by chance, the higher the score on this axis the lower the probability the association between gene and pathway is by chance alone. The strongest association revealed by the analysis is between genes altered in POUND mice and those associated with IGF-1 signaling. The open blue boxes connected by the lines represent ratio values indicating the ratio of genes detected in the pathway to the total number of genes in that particular pathway. C. Heat map from reverse phase protein analysis comparing protein expression in hindlimb muscle of POUND mice with that of normal mice. Arrows indicate proteins including Akt, MAPK, and MEK that are highly expressed in muscle from normal mice (red) but not highly expressed in muscle from POUND mice (green). Western blots shown on the right are for total and phosphorylated Akt, MAPK, and MEK.</p

Primary myoblasts from POUND mice show impaired proliferation and differentiation.

<p>A. Primary myoblasts cultures from wild-type and POUND mice show a significant decrease in the proliferation and metabolic activity of myoblasts in POUND mice compared to normal wild-type mice as measured using MTS assay (left panel). B. Myoblasts from POUND mice (right panel, top micrograph) fail to differentiate normally and after 7 days do not develop into the elongate myotubes characteristic of normal, wild-type mice (right panel, bottom micrograph). C. Real-time PCR data show that that the early marker of myoblast differentiation, MyoD (left graph), and the later differentiation marker myogenin (right graph) are both significantly downregulated in myoblasts from POUND mice.</p

Relative muscle cross-sectional area and fiber number (mean, S.D.) for the hindlimb muscles of POUND and WT mice.

<p>Fiber number is calculated by dividing muscle CSA by the average fiber cross-sectional area.</p

Leptin increases myoblast proliferation.

<p>A. Leptin-treatment (100 ng/ml) significantly increases cell proliferation and metabolic activity measured using MTS assay in primary myoblasts from mice 12 months of age. B. Leptin-treatment (100 ng/ml) also significantly increases cell proliferation and metabolic activity measured using MTS assay in primary myoblasts from mice 24 months of age. C. -treatment (100 ng/ml) significantly increases the expression of the myogenic factors MyoD and myogenin in primary myoblasts from mice 24 months of age. Leptin did not increase the expression of these factors in myoblasts from mice 12 months of age. D. Box-and-whisker plots showing ΔΔCt values (y-axis) for leptin (LEP) and leptin receptor (LEPR) expression in the soleus (SOL; top row) and extensor digitorum longus (EDL; bottom row) muscles of mice 12 and 24 months of age (x-axis). The whiskers mark the minimum and maximum values, the boxes the first and third quartiles, and the bar within the box indicates the median. Expression of the leptin receptor is not increased with age, and is significantly (P<.05) downregulated in aged soleus (SOLLEPR).</p

Pharmacological TLR4 inhibition does not affect fear-learning and memory.

<p>(<b>A</b>) aCSF infused mice (n = 10) show similar association curves in the fear-conditioning paradigm compared with TLR4 antagonist infused mice (n = 10). (<b>B</b>) aCSF infused mice (n = 10) show similar freezing levels in the fear-conditioning paradigm compared with TLR4 antagonist infused mice (n = 10). (<b>C</b>) Average freezing during contextual fear. (<b>D</b>) aCSF infused mice (n = 10) show similar freezing in the fear-conditioning paradigm compared with TLR4 antagonist infused mice (n = 10) in the presence of tone.</p

CREB and p-CREB are upregulated in the hippocampus of TLR4^−/− mice.

<p>Brains from TLR4^+/+ (n = 8) and TLR4^−/− (n = 8) mice were dissected and hippocampi were removed. Tissues were then lysed, electrophoresed and immunoblotted against GluR1, CREB, ERK and their phosphorylated forms. Representative blots demonstrate that levels of CREB and pCREB were upregulated in TLR4^−/− mice compared to TLR4^+/+ mice, whereas GluR1, ERK and their phosphorylated forms were not changed. * p<0.05.</p

Developmental TLR4 deficiency, but not pharmacological TLR4 antagonism, enhances retention of spatial reference memory.

<p>(<b>A</b>) TLR4^−/− (n = 19) and TLR4^+/+ (n = 24) mice were tested in probe trials at 24, 48, 72 and 96 hours following training for retention of spatial reference memory. Tests were done after all experimental groups exhibited loss of memory of the platform location. Mean distance from the platform was measured and used to indicate efficiency in locating the hidden platform. TLR4^−/− mice showed shorter mean distance from the platform at 24 and 48 hours after training compared with TLR4^+/+ mice, indicating a more accurate swim toward the platform quadrant (<b>B</b>) Mice (C57BL/6) were implanted with an osmotic pump containing either aCSF (n = 10) or a TLR4 antagonist (n = 10). The pump was connected via tubing to a cannula, which was positioned to the lateral ventricle. Following training in the MWM task, mice were tested in probe trials at 24 and 48 hours following training for retention of spatial reference memory. Both experimental groups exhibited similar performance during probe trials, as measured by mean distance from the platform.</p

Developmental TLR4 deficiency impairs fear-learning and memory.

<p>(<b>A</b>) TLR4^−/− (n = 19) mice show impaired association curves in the fear-conditioning paradigm compared with TLR4^+/+ (n = 24) mice. (<b>B</b>) TLR4^−/− mice exhibit significantly impaired hippocampus dependent contextual fear compared to TLR4^+/+ mice, as measured by time freezing during 5 minutes of exposure to the original context. (<b>C</b>) Average freezing during contextual fear. (<b>D</b>) TLR4^−/− mice exhibit reduced freezing compared with TLR4^+/+ mice in the presence of tone, indicating impaired fear response.</p