The delaying effect of alpha-glycerophosphocholine on senescence, transthyretin deposition, and osteoarthritis in senescence-accelerated mouse prone 8 mice

<p>Administration of alpha-glycerophosphocholine (GPC), a choline compound in food, is expected to contribute to human health. In this study, we evaluated its effect on aging in senescence-accelerated mouse prone 8 (SAMP8) mice. Male SAMP8 mice had free access to a commercial stock diet and drinking water with or without GPC (0.07 mg/ml). Mice in the GPC group had significantly lower total senescence grading score than that of the control group at 36 weeks of age. Administration of GPC decreased the deposition of transthyretin (TTR), an amyloidogenic protein, in the brain. Aggregated TTR activated microglia and led to neuroinflammation. Thus, GPC would protect the brain by reducing TTR deposition and preventing neuroinflammation. In a histological study of knee joints, it was found that SAMP8 mice administered GPC showed decreased joint degeneration. These results suggest that GPC delays the aging process and may be a useful compound in anti-aging functional food development.</p> <p>Transthyretin (TTR) deposition was reduced by administration of alpha-glycerophosphocholine in senescence-accelerated mouse prone 8 mice.</p

Data_Sheet_1_AMP-Activated Protein Kinase Mediates the Effect of Leptin on Avian Autophagy in a Tissue-Specific Manner.DOCX

<p>Autophagy, a highly conserved intracellular self-digestion process, plays an integral role in maintaining cellular homeostasis. Although emerging evidence indicate that the endocrine system regulates autophagy in mammals, there is still a scarcity of information on autophagy in avian (non-mammalian) species. Here, we show that intracerebroventricular administration of leptin reduces feed intake, modulates the expression of feeding-related hypothalamic neuropeptides, activates leptin receptor and signal transducer and activator of transcription (Ob-Rb/STAT) pathway, and significantly increases the expression of autophagy-related proteins (Atg3, Atg5, Atg7, beclin1, and LC3B) in chicken hypothalamus, liver, and muscle. Similarly, leptin treatment activates Ob-Rb/STAT pathway and increased the expression of autophagy-related markers in chicken hypothalamic organotypic cultures, muscle (QM7) and hepatocyte (Sim-CEL) cell cultures as well as in Chinese Hamster Ovary (CHO-K1) cells-overexpressing chicken Ob-Rb and STAT3. To define the downstream mediator(s) of leptin's effects on autophagy, we determined the role of the master energy sensor AMP-activated protein kinase (AMPK). Leptin treatment significantly increased the phosphorylated levels of AMPKα1/2 at Thr172 site in chicken hypothalamus and liver, but not in muscle. Likewise, AMPKα1/2 was activated by leptin in chicken hypothalamic organotypic culture and Sim-CEL, but not in QM7 cells. Blocking AMPK activity by compound C reverses the autophagy-inducing effect of leptin. Together, these findings indicate that AMPK mediates the effect of leptin on chicken autophagy in a tissue-specific manner.</p

Data_Sheet_2_AMP-Activated Protein Kinase Mediates the Effect of Leptin on Avian Autophagy in a Tissue-Specific Manner.DOCX

<p>Autophagy, a highly conserved intracellular self-digestion process, plays an integral role in maintaining cellular homeostasis. Although emerging evidence indicate that the endocrine system regulates autophagy in mammals, there is still a scarcity of information on autophagy in avian (non-mammalian) species. Here, we show that intracerebroventricular administration of leptin reduces feed intake, modulates the expression of feeding-related hypothalamic neuropeptides, activates leptin receptor and signal transducer and activator of transcription (Ob-Rb/STAT) pathway, and significantly increases the expression of autophagy-related proteins (Atg3, Atg5, Atg7, beclin1, and LC3B) in chicken hypothalamus, liver, and muscle. Similarly, leptin treatment activates Ob-Rb/STAT pathway and increased the expression of autophagy-related markers in chicken hypothalamic organotypic cultures, muscle (QM7) and hepatocyte (Sim-CEL) cell cultures as well as in Chinese Hamster Ovary (CHO-K1) cells-overexpressing chicken Ob-Rb and STAT3. To define the downstream mediator(s) of leptin's effects on autophagy, we determined the role of the master energy sensor AMP-activated protein kinase (AMPK). Leptin treatment significantly increased the phosphorylated levels of AMPKα1/2 at Thr172 site in chicken hypothalamus and liver, but not in muscle. Likewise, AMPKα1/2 was activated by leptin in chicken hypothalamic organotypic culture and Sim-CEL, but not in QM7 cells. Blocking AMPK activity by compound C reverses the autophagy-inducing effect of leptin. Together, these findings indicate that AMPK mediates the effect of leptin on chicken autophagy in a tissue-specific manner.</p

Data_Sheet_4_AMP-Activated Protein Kinase Mediates the Effect of Leptin on Avian Autophagy in a Tissue-Specific Manner.DOCX

<p>Autophagy, a highly conserved intracellular self-digestion process, plays an integral role in maintaining cellular homeostasis. Although emerging evidence indicate that the endocrine system regulates autophagy in mammals, there is still a scarcity of information on autophagy in avian (non-mammalian) species. Here, we show that intracerebroventricular administration of leptin reduces feed intake, modulates the expression of feeding-related hypothalamic neuropeptides, activates leptin receptor and signal transducer and activator of transcription (Ob-Rb/STAT) pathway, and significantly increases the expression of autophagy-related proteins (Atg3, Atg5, Atg7, beclin1, and LC3B) in chicken hypothalamus, liver, and muscle. Similarly, leptin treatment activates Ob-Rb/STAT pathway and increased the expression of autophagy-related markers in chicken hypothalamic organotypic cultures, muscle (QM7) and hepatocyte (Sim-CEL) cell cultures as well as in Chinese Hamster Ovary (CHO-K1) cells-overexpressing chicken Ob-Rb and STAT3. To define the downstream mediator(s) of leptin's effects on autophagy, we determined the role of the master energy sensor AMP-activated protein kinase (AMPK). Leptin treatment significantly increased the phosphorylated levels of AMPKα1/2 at Thr172 site in chicken hypothalamus and liver, but not in muscle. Likewise, AMPKα1/2 was activated by leptin in chicken hypothalamic organotypic culture and Sim-CEL, but not in QM7 cells. Blocking AMPK activity by compound C reverses the autophagy-inducing effect of leptin. Together, these findings indicate that AMPK mediates the effect of leptin on chicken autophagy in a tissue-specific manner.</p

Data_Sheet_3_AMP-Activated Protein Kinase Mediates the Effect of Leptin on Avian Autophagy in a Tissue-Specific Manner.DOCX

<p>Autophagy, a highly conserved intracellular self-digestion process, plays an integral role in maintaining cellular homeostasis. Although emerging evidence indicate that the endocrine system regulates autophagy in mammals, there is still a scarcity of information on autophagy in avian (non-mammalian) species. Here, we show that intracerebroventricular administration of leptin reduces feed intake, modulates the expression of feeding-related hypothalamic neuropeptides, activates leptin receptor and signal transducer and activator of transcription (Ob-Rb/STAT) pathway, and significantly increases the expression of autophagy-related proteins (Atg3, Atg5, Atg7, beclin1, and LC3B) in chicken hypothalamus, liver, and muscle. Similarly, leptin treatment activates Ob-Rb/STAT pathway and increased the expression of autophagy-related markers in chicken hypothalamic organotypic cultures, muscle (QM7) and hepatocyte (Sim-CEL) cell cultures as well as in Chinese Hamster Ovary (CHO-K1) cells-overexpressing chicken Ob-Rb and STAT3. To define the downstream mediator(s) of leptin's effects on autophagy, we determined the role of the master energy sensor AMP-activated protein kinase (AMPK). Leptin treatment significantly increased the phosphorylated levels of AMPKα1/2 at Thr172 site in chicken hypothalamus and liver, but not in muscle. Likewise, AMPKα1/2 was activated by leptin in chicken hypothalamic organotypic culture and Sim-CEL, but not in QM7 cells. Blocking AMPK activity by compound C reverses the autophagy-inducing effect of leptin. Together, these findings indicate that AMPK mediates the effect of leptin on chicken autophagy in a tissue-specific manner.</p

Discovery of the Elusive Leptin in Birds: Identification of Several ‘Missing Links’ in the Evolution of Leptin and Its Receptor

<div><p>Leptin is a pleiotropic protein best known for regulation of appetite and fat storage in mammals. While many leptin orthologs have been identified among vertebrates, an authentic leptin in birds has remained elusive and controversial. Here we identify leptin sequence from the Peregrine falcon, <i>Falco peregrinus</i> (pfleptin), and identify sequences from two other birds (mallard and zebra finch), and ‘missing’ vertebrates (elephant shark, alligator, Indian python, Chinese soft-shelled turtle, and coelacanth). The pattern of genes surrounding <i>leptin</i> (<i>snd1</i>, <i>rbm28</i>) is syntenic between the falcon and mammalian genomes. Phylogenetic analysis of all known leptin protein sequences improves our understanding of leptin’s evolution. Structural modeling of leptin orthologs highlights a highly conserved hydrophobic core in the four-helix cytokine packing domain. A docked model of leptin with the leptin receptor for Peregrine falcon reveals several conserved amino acids important for the interaction and possible coevolution of leptin with its receptor. We also show for the first time, an authentic avian leptin sequence that activates the JAK-STAT signaling pathway. These newly identified sequences, structures, and tools for avian leptin and its receptor will allow elucidation of the function of these proteins in feral and domestic birds.</p></div

Bird Leptin gene and protein product compared to other vertebrate species.

<p><b>A)</b> Phylogenetic analysis using Maximum Likelihood analysis of selected vertebrate species placing peregrine falcon and zebra finch leptin protein (red) most similar to alligator. <b>B)</b><i>Leptin</i> synteny in both Peregrine and Saker falcon show chromosomal location similar to that of mouse and human. Distances for the avian species are much closer than in mouse or human, while DNA sequence for the entire region analyzed contains a higher GC content.</p

Peregrine falcon leptin model.

<p><b>A)</b> Structural model of Peregrine falcon leptin showing conserved (red) or variant (gray) amino acids between human and peregrine falcon. <b>B)</b> Molecular dynamic simulations for 10 nanoseconds (ns) of various leptin models including the falcon (black). Plots are shown as the average movement of the carbon alpha backbone at each step in time. <b>C)</b> The carbon alpha RMSD showing the molecular movement to stabilize in the first few nanoseconds and stay around 3Å over the entire simulation. <b>D)</b> Average movement of each amino acid over the entire simulation shows stability of the four helices with higher dynamics in the loops.</p

Purification of Peregrine falcon lepin (pfleptin).

<p><b>A)</b> Coomassie stain following 15% Tris-Tricine SDS PAGE. Control lysate and lysate of the pJ414 pfleptin expressing cells were previously passed over Ni-Sepharose and dialyzed into 125 mM NaCl. 15 μL of each sample were loaded onto the gel. The pfleptin protein with tag is 17 kDa. <b>B)</b> Western blot of A following transfer to membrane and probed with an anti-His primary antibody. <b>C)</b> Glutathione pull down of the pGEX4T-chLepR(227–628) which can be seen at 71 kDa. Beads were then incubated with either the control lysate or the pfleptin lysate. This resulted in concentrating the pfleptin protein at 17 kDa. <b>D)</b> Luciferase assays showing activation of the STAT pathway of chicken leptin receptor transfected CHO cells treated with concentrated peregrine falcon leptin (n  =  2). Error bars represent the standard error of the mean and * represents a p-value ≤ 0.005 between samples. No significance was seen between the two concentrations of leptin treatment suggesting maximum saturation in the assay was reached.</p

Sequences used in structurally modeling Leptin proteins throughout vertebrate evolution.

<p><b>A)</b> Amino acids that are conserved in all 16 models shown (red) on the known structure of human leptin, with the four helices identified. The four helix bundle is conserved in all species through hydrophobic amino acids. <b>B)</b> Z-score for each model as determined using the YASARA2 force field. Values between 0 and −2 are considered to be good models. <b>C)</b> Structural alignment of each of energy minimized models to the human structure showing the average carbon alpha RMDS in angstroms (Å).</p