MOESM1 of Advanced glycation end products induce brain-derived neurotrophic factor release from human platelets through the Src-family kinase activation

Additional file 1: Figure S1. Scatter plot of BDNF levels in whole blood, serum and platelets (1 × 108 cells) (n = 10). Table S1. The blood information of the volunteers. The blood samples were sent to SRL Inc, Japan. and fasting plasma glucose (FPG), fasting immunoreactive insulin (FIRI), HbA1c was analyzed at SRL. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated from FIRI and FPG by the following equation: HOMA-R = FIRI (mU/l) × FPG (mmol/l)/22.5. Data are means ± SEM. Figure S2. BDNF release at higher AGE concentrations (100 μg–1 mg/ml AGE). Data are presented as means ± SEM (n = 8). Statistical analyses were performed by SPSS one-way ANOVA. *p < 0.05 vs control. Note that 100 mg/ml was the saturation dose. Figure S3. Time course of AGE-induced BDNF release. BDNF release was measured at 5, 20 and 60 min after AGE stimulation. White bar, control; light grey bar, 25 μg/ml AGE; dark grey bar, 50 μg/ml AGE; black bar, 100 μg/ml AGE (n = 8, means ± SEM). Statistical analysis was performed by one-way ANOVA. *p < 0.05 vs control. The graph at 5 min is same as Fig. 2. Figure S4. AGE induced PF4 and 5-HT release from human platelets. Data were presented as means ± SEM (n = 8). Statistical analysis were performed by SPSS paired t test. White bars, control; black bars, AGE (100 μg/ml). *p < 0.05 vs control. Figure S5. AGE increased intracellular Ca2+ levels in platelets. Platelet was incubated with Oregon Green 488 (2 mM, Thermo Fisher) for 30 min and was washed with assay buffer (same as BDNF assay buffer). (a), Fluorescent intensity was measured by fluorescent microplate reader (Ex: 490 nm, EM: 530 nM) 3 min after AGE stimulation according to the manufacture’s instruction. Data are presented as means ± SEM (n = 30 (well)). Typical fluorescent photomicrographs of control (b) and AGE-treated (c) platelets

Attenuated hepatic inflammation and liver injury in mice fed AHF supplemented with EPA or DHA.

<p>Hematoxylin and eosin (H&E) staining of liver sections from representative mice from each treatment group (A), and plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (B). n = 11–15 per group. ** p < 0.01, *** p < 0.001 versus chow group; # p < 0.05, ### p < 0.001 versus AHF group.</p

Effect of EPA and DHA on hepatic lipid content in mice fed AHF diet.

<p>Liver macroscopic picture (A) and hepatic triglyceride (TG) and total cholesterol (T-Cho) levels (B) in mice fed a normal chow, an AHF diet, or an AHF diet supplemented with EPA or DHA for four weeks. n = 12–15 per group. ** p < 0.01, *** p < 0.001 versus chow group; ## p < 0.01, ### p < 0.001 versus AHF group.</p

Effect of AHF diet supplemented with EPA or DHA on hepatic fibrogenesis.

<p>Quantitative real-time PCR of genes involved in fibrogenesis in the livers of mice fed a normal chow, an AHF diet, or an AHF diet supplemented with EPA or DHA for four weeks (A). Immunoblot analysis of α-SMA levels and the ratio between α-SMA and β-actin by densitometry analysis (B). n = 13–15 per group. * p < 0.05 versus chow group; # p < 0.05 versus AHF group.</p

Effect of AHF diet supplemented with EPA or DHA on hepatic oxidative stress.

<p>Representative immunohistochemical staining for Nε-(Hexanoyl) Lysine in liver section form mice fed a normal chow, an AHF diet, or an AHF diet supplemented with EPA or DHA for four weeks (A). Liver 8-OHdG levels in mice fed a normal chow, an AHF diet, or an AHF diet supplemented with EPA or DHA for four weeks (B). Quantitative real-time PCR of genes involved in oxidative stress in the livers of mice fed a normal chow, an AHF diet, or an AHF diet supplemented with EPA or DHA for four weeks (B). n = 13–15 per group. * p < 0.05 versus chow group; # p < 0.05 versus AHF group.</p

Hepatic fatty acid composition in mice fed normal chow, AHF, AHF + EPA and AHF + DHA diets for four weeks.

<p>Hepatic fatty acid composition (A) and the n-6/n-3 fatty acid ratio (B) in livers of mice fed a normal chow, an AHF diet, or an AHF diet supplemented with EPA or DHA for four weeks. n = 3–4 per group. * p < 0.05, *** p < 0.001 versus chow group; # p < 0.05, ### p < 0.001 versus AHF group.</p

Phenotypic comparison of C57BL/6J mice fed the chow, AHF, AHF + EPA, and AHF + DHA diet for 4 weeks.

<p>Phenotypic comparison of C57BL/6J mice fed the chow, AHF, AHF + EPA, and AHF + DHA diet for 4 weeks.</p

Effect of AHF diet supplemented with EPA or DHA on hepatic lipid metabolism-related mRNA and protein levels.

<p>For gene expression and immunoblot analyses, livers were collected from the mice fed a normal chow, an AHF diet, or an AHF diet supplemented with EPA or DHA for four weeks. Quantitative real-time PCR of genes involved in fatty acid and TG synthesis (A), fatty acid oxidation (C), PUFA synthesis (D), lipid storage (E), cholesterol and lipoprotein metabolism (F). Immunoblot analysis for mature SREBP-1 levels (B). n = 13–15 per group. * p < 0.05 versus chow group; # p < 0.05 versus AHF group.</p

Effect of AHF diet supplemented with EPA or DHA on hepaticinflammation.

<p>Representative immunohistochemical staining for F4/80 in liver section form mice fed a normal chow, an AHF diet, or an AHF diet supplemented with EPA or DHA for four weeks (A). Quantitative real-time PCR of genes involved in inflammation in the livers of mice fed a normal chow, an AHF diet, or an AHF diet supplemented with EPA or DHA for four weeks (B). Immunoblot analysis of phosphorylated JNK and total JNK levels and the ratio between phosphorylated and total JNK by densitometry analysis (C). n = 13–15 per group. * p < 0.05 versus chow group; # p < 0.05 versus AHF group.</p

Single-cell transcriptome profiling of pancreatic islets from early diabetic mice identifies Anxa10 for Ca2+ allostasis toward β-cell failure

Type 2 diabetes (T2D) is a progressive disorder denoted by hyperglycemia and impaired insulin secretion. Although a decrease in β-cell function and mass is a well-known trigger for diabetes, the comprehensive mechanism is still unidentified. Here we carried out single-cell RNA sequencing (scRNA-seq) of pancreatic islets from prediabetic and diabetic db/db mice, an animal model of T2D. We discovered a diabetes-specific transcriptome landscape of endocrine and nonendocrine cell types with subpopulations of β and α cells. We recognized a new prediabetic gene, Anxa10, that was induced by, and regulated Ca2+ influx from metabolic stresses. Anxa10-overexpressed β cells displayed suppression of glucose-stimulated intracellular Ca2+ elevation and potassium-induced insulin secretion. Pseudotime analysis of β cells predicted this Ca2+-surge responder-cluster proceeded to mitochondria dysfunction and also endoplasmic reticulum stress. Other trajectories comprised dedifferentiation and transdifferentiation, emphasizing acinar-like cells in diabetic islets. Altogether, our data provides a new insight into Ca2+ allostasis and β cell failure processes.</p