APN induces autophagy in macrophage through Akt-FOXO3a pathway.

<p>A, Western blot shows the protein level of p-Akt, Akt, p-FOXO3a, FOXO3a in macrophages stimulated with phosphate buffered saline, APN, Akt agonist (740Y-P) or with APN in combination with 740Y-P. B, Western blot shows the protein level of PTEN, p-mTOR, mTOR in macrophages stimulated with phosphate buffered saline, APN, Akt agonist (740Y-P) or with APN in combination with 740Y-P. C, Quantitative analysis of p-Akt/Akt ratio, p-FOXO3a/FOXO3a ratio and p-mTOR/mTOR ratio in macrophages stimulated with phosphate buffered saline, APN, and APN in combination with 740Y-P, respectively. n = 6 per group. *P<0.05 versus macrophage treated with saline. D, Quantification of the optical density of PTEN in each groups. n = 6 per group. *P<0.05 versus macrophage treated with saline.</p

APN exacerbates macrophage autophagy in vitro.

<p>A and B respectively show the representative western blot and quantitative analysis of LC3 protein level in VSMC and macrophage stimulated with or without APN (5 μg/ml). n = 6 per group. *P<0.05 versus macrophage without APN. C and D respectively show the western blot and quantitative analysis of P62 and Beclin 1 protein level in macrophage treated with or without APN (5 μg/ml). n = 6 per group. *P<0.05 versus macrophage without APN.</p

APN secreted by PVAT aggravates autophagy in plaque.

<p>A, Representative western blot of LC3 expression in arteria carotis transplanted with WT or APN^-/- PVAT 4 weeks after atherosclerosis (n = 6 per group). B, Quantitative analysis of LC3 protein expression in various groups. *P<0.05 versus (WT) PVAT. C, Immunofluorescence of p62, another marker of autophagy, in the arteria carotis with WT or APN^-/- PVAT (n = 6 per group). D, Histogram shows p62 positive cells per 100 cells. *P<0.05 versus (WT) PVAT.</p

Differential expression of genes identified by suppression subtractive hybridization in liver and adipose tissue of gerbils with diabetes

<div><p>Objectives</p><p>We aimed at identifying genes related to hereditary type 2 diabetes expressed in the liver and the adipose tissue of spontaneous diabetic gerbils using suppression subtractive hybridization (SSH) screening.</p><p>Methods</p><p>Two gerbil littermates, one with high and the other with normal blood glucose level, from our previously bred spontaneous diabetic gerbil strain were used in this study. To identify differentially expressed genes in the liver and the adipose tissue, mRNA from these tissues was extracted and SSH libraries were constructed for screening. After sequencing and BLAST analyzing, up or down-regulated genes possibly involved in metabolism and diabetes were selected, and their expression levels in diabetic gerbils and normal controls were analyzed using quantitative RT-PCR and Western blotting.</p><p>Results</p><p>A total of 4 SSH libraries were prepared from the liver and the adipose tissue of gerbils. There are 95 up or down-regulated genes were identified to be involved in metabolism, oxidoreduction, RNA binding, cell proliferation, and differentiation or other function. Expression of 17 genes most possibly associated with diabetes was analyzed and seven genes (<i>Sardh</i>, <i>Slc39a7</i>, <i>Pfn1</i>, <i>Arg1</i>, <i>Cth</i>, <i>Sod1</i> and <i>P4hb</i>) in the liver and one gene (<i>Fabp4</i>) in the adipose tissue were identified that were significantly differentially expressed between diabetic gerbils and control animals.</p><p>Conclusions</p><p>We identified eight genes associated with type 2 diabetes from the liver and the adipose tissue of gerbils via SSH screening. These findings provide further insights into the molecular mechanisms of diabetes and imply the value of our spontaneous diabetic gerbil strain as a diabetes model.</p></div

Classification of genes from suppression subtractive hybridization (SSH) libraries.

<p>(A) Putative functional classification of 78 genes from the liver SSH library for which identity was could be inferred. Information on function from previous reports or from the Mouse Genome Informatics database. These genes were distributed into 11 groups and were non-annotated based on their functions. (B) Seventeen known genes from the adipose SSH library were divided into seven groups according to their function. Methods were the same as in A.</p

Gene names, primer sequences, annealing temperatures, and lengths of PCR products for qPCR analysis of 17 genes and two housekeeping genes (as referencing controls) related to diabetes and metabolism identified from SSH libraries.

<p>Gene names, primer sequences, annealing temperatures, and lengths of PCR products for qPCR analysis of 17 genes and two housekeeping genes (as referencing controls) related to diabetes and metabolism identified from SSH libraries.</p

mRNA expression levels of six candidate genes selected from two SSH libraries from the liver and adipose tissue of gerbils.

<p>(A-E) Relative mRNA expression levels of five genes (<i>Sardh</i>, <i>Pfn1</i>, <i>P4hb</i>, <i>Slc39a7</i> and <i>Sod1</i>) between control and diabetic gerbils in the liver. (F) Relative mRNA expression levels of <i>Fabp4</i> between control and diabetic gerbils in adipose tissue. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

The percentages of animals with FPG≥5.2, PG2h≥6.8 and both FPG≥5.2and PG2h≥6.8 (mmol/l).

<p>Animals of FPG≥5.2, PG2h≥6.8 and both FPG≥5.2and PG2h≥6.8 were increased with the number of breeding generations from F0 to F6.</p

The pathological analysis of target organs between control group and experimental group using HE stain (100×, 400×): liver (A), kidney (B), and pancreas (C).

<p>(A): in liver, we can find severe hepatic steatosis and focal necrosis in diabetic <i>gerbils</i> was the liver pathological changes.; (B): the kidney are glomerulus atrophy and the tubular can see protein substance, also some of the nucleus pycnosis, cataclastic, and solution in the end in diabetic <i>gerbils</i>; (C): the pancreas represent nuclear pyrosis and cell necrosis leading to local necrosis in diabetes <i>gerbil</i>. All of these three organs had histological changes between control and experimental group.</p

Expression level of the diabetic related genes between control and experimental gerbils with western blot and RT-qPCR.

<p>(A) was the <i>adiponectin</i> expression changes at protein and mRNA level, both of them were significantly lower in high blood glucose group than that in control group; (B) was the <i>letpin</i> expression change at protein level was significantly higher in high blood glucose group than that in control group; (C) was the <i>CAPN10</i> expression change at mRNA level, it had a tendency of lower expression in high blood glucose group. Notes: “*” (<i>p</i>≤0.05), “**” (<i>p</i>≤ 0.01), and “***” (<i>p</i>≤0.001) showed significantly different between experimental and control animals.</p