The contents of the autophagic proteins in the hypothalamus from diet induced obese mice with 8 and 16 weeks of treatment.

<p><b>A.</b> Western-blotting shows the Beclin-1 to β-actin ratio in the hypothalamus of obese mice with 8 weeks on a HFD. <b>B.</b> Western-blotting shows LC3-II to β-actin ratio in the hypothalamus of obese mice with 8 weeks on a HFD. <b>C.</b> p62 content in the hypothalamus of obese mice with 8 weeks on a HFD. <b>D.</b> Beclin-1 content in the hypothalamus of obese mice with 16 weeks on a HFD. <b>E.</b> LC3-II to β-actin ratio in the hypothalamus of obese mice with 16 weeks on a HFD. <b>F.</b> p62 content in the hypothalamus of obese mice with 16 weeks on a HFD. Values are shown as mean ± SEM. *P < 0.05; n = 4–7 animals per group.</p

Rapamycin treatment decreases inflammation, endoplasmic reticulum stress and apoptosis markers in obese mice.

<p><b>A and B.</b> Western blotting of pJNK and pEIF2α content in the hypothalamus of obese mice (16 weeks on a HFD) treated (once per day) for 5 days with 2 μl of a solution that contained 25 μM rapamycin (Rap) or saline (Sal). <b>C and D.</b> Bax and Bcl-2 content in hypothalamus of obese mice treated with rapamycin. Values are shown as mean ± SEM. *P < 0.05; n = 5–6 animals per group.</p

mRNA gene expression of autophagic proteins in the hypothalamus of obese mice.

<p>QRT-PCR was used to analyze the gene expression of <b>A.</b> Ulk1, <b>B.</b> BECN-1, <b>C.</b> ATG5, <b>D.</b> Map1lc3 and <b>E.</b> SQSTM1 in the hypothalamus of diet induced obese mice (16 weeks on a HFD) versus lean mice. Values are shown as mean ± SEM. *P < 0.05; n = 6 animals per group.</p

Effect of saturated fatty acid in a neuron cell line.

<p><b>E</b>. Western-blotting shows the Beclin-1 and p62 to β-actin ratio, respectively in CLU-189 cell line treated with 500 μM of palmitic acid for 6, 12 or 24 hours. Values are shown as mean ± SEM. *P < 0.05;</p

Metabolic parameters in mice treated with Rapamycin (icv).

<p><b>A</b> and <b>B</b>. GTT curve and area under the GTT of lean or obese mice (16 weeks of a HFD) treated with 2 μl of a solution that contained 25 μM rapamycin (Rap) or saline (Sal) once a day for 6 days. <b>C and D.</b> Food intake and weight modulation in obese mice treated with rapamycin. HFD versus chow diet in mice treated with saline and a HFD in mice treated with rapamycin versus a HFD in mice treated with saline. Values are shown as mean ± SEM. *P < 0.05; n = 5–6 animals per group.</p

Rapamycin treatment reverses the autophagic downregulation in obese mice.

<p><b>A.</b> Western blotting showing LC3-II to β-actin ratio in the hypothalamus of diet induced obese mice (16 weeks on a HFD) treated (once per day) for 5 days with 2 μl of a solution that contained 25 μM rapamycin (Rap) or saline (Sal). <b>B.</b> The p62 content in the hypothalamus of the diet induced obese mice treated with rapamycin. Values are shown as mean ± SEM. *P < 0.05; n = 5–6 animals per group.</p

Effect of treatment with conditioned medium on hypothalamic neuron cell lien.

<p><b>A, B, C and D.</b> QRT-PCR was used to analyze the gene expression of TNF, Map1lc3, Sqstm1, Becn1, respectively. <b>E</b>. Western-blotting shows the Beclin-1, LC3-II and p62 to β-actin ratio, respectively in CLU-189 cell line treated with conditioned medium of microglia cells BV-2 treated with 500 μM of palmitic acid for 6 hours. Values are shown as mean ± SEM. *P < 0.05;</p

Weight gain and inflammatory markers in hypothalamus of obese mice.

<p><b>A.</b> Weight gain of mice fed a high fat diet compared to chow for 16 weeks. QRT-PCR was used to analyze the gene expression of <b>B.</b> IL1-β, <b>C.</b> TNFR1 and <b>D.</b> IL-10 in the hypothalamus of DIO mice at 8 weeks of treatment. QRT-PCR was used to analyze the gene expression of <b>E.</b> IL1-β, <b>F.</b> TNFR1 and <b>G.</b> IL-10 in the hypothalamus of DIO mice at 16 weeks of treatment. Values are shown as mean ± SEM. *P < 0.05; n = 6 animals per group.</p

Experimental protocol of metabolic programming used in the study.

<p>Dams were fed with standard chow (SC) or a high-fat diet (HFD) during the adaptation period, pregnancy and lactation. After weaning, offspring was fed only with SC until d28 or d42. After d42, offspring from obese (H) or control dams (C) received SC (CC and HH) or HFD (CC-HF and HH-HF) until d82.</p

Antibodies used in Western Blot analysis.

<p>Antibodies used in Western Blot analysis.</p