Effect of CMCC on metabolic-related genes in muscle of young broiler chicks.

<p>Relative expression of mitochondrial- and metabolic-related genes was determined by qPCR (a). Phosphorylated and total protein levels of AMPKα1/α2 and mTOR were determined by Western blot (b) and presented as p-protein/total protein ratio (c). The values represent the mean ±SEM (n = 6). * Indicates a significant difference between cold and control group (<i>P</i><0.05).</p

Effect of CMCC on HSP and HSF expression in the muscle of young broiler chicks.

<p>HSP and HSF mRNA levels were measured by qPCR (a). HSP70 and HSP90 protein levels were determined by Western blot (b) and presented as normalized ratio to β-actin (c). The values represent the mean ± SEM (n = 6). *Indicates a significant difference between cold and control group (<i>P</i><0.05).</p

Effect of CMCC on lipogenesis-related genes in liver of young broiler chicks.

<p>Relative expression of lipogenic genes and their related transcription factors (a) was measured by qPCR as described in material and methods. Phosphorylated and total protein levels of AMPK, mTOR and ACC were determined by Western blot (b) and presented as p-protein/total protein ratio (c). β-actin was used as loading and housekeeping control. Data are presented as mean ± SEM (n = 6). *Indicate a significant difference between cold and control group (<i>P</i><0.05).</p

Effect of CMCC on plasma metabolite levels.

<p>Plasma levels of Chol, Glc, TG, UA (a), LDH and CK (b) were determined at the first week using commercial kits as described in materials and methods. Data are presented as mean ± SEM (n = 6). *Indicate a significant difference between cold and control group (<i>P</i><0.05).</p

Effect of CMCC on feeding-related genes and HSPs in the brain of young broiler chicks.

<p>Relative expression of hypothalamic feeding-related neuropeptides (a), lipogenic genes (b), HSPs and HSF (c) were determined by qPCR using 2^-ΔΔCt method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142319#pone.0142319.ref025" target="_blank">25</a>]. Data are presented as mean ± SEM (n = 6). * Indicate a significant difference between cold and control group (<i>P</i><0.05).</p

Effect of CMCC on growth performances in young broiler chicks.

<p>Cumulative feed intake FI (a), body weight gain BWG (b), feed conversion ratio FCR (c), and body temperature (d). Data are presented as mean ± SEM (n = 40) for each week and for the total experimental period (3 weeks). *<i>P</i><0.05. Different letters indicate daily difference in body temperature within each group (a-c and α, difference within control and cold group, respectively).</p

Effect of CMCC on HSP and HSF expression in liver of young broiler chicks.

<p>HSP and HSF mRNA levels were measured by qPCR (a). HSP70 and HSP90 protein levels were determined by Western blot (b) and presented as normalized ratio to β-actin (c). The values represent the mean ± SEM (n = 6). *Indicates a significant difference between cold and control group (<i>P</i><0.05).</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