Effect of supplementation with powdered persimmon leaf on hepatic oxidative stress in C57BL/KsJ-<i>db/db</i> mice.

<p>A; hepatic TBARS level. B; hepatic mitochondria hydrogen peroxide content. C; hepatic antioxidant enzyme activity. D; hepatic antioxidant gene expression. The mRNA levels were analyzed by Northern blot analysis using SOD, CAT and GPX probes and normalized to an internal control (GAPDH). Three independent analyses were performed. Values are the mean ± S.E., n = 10. *p<0.05, **p<0.01, ***p<0.001. CAT; catalase, GPX; glutathione peroxidase, GAPDH; glyceraldehyde-3-phosphate dehydrogenase, PL; powdered persimmon leaf, SOD; superoxide dismutase.</p

Opposite Expression of SPARC between the Liver and Pancreas in Streptozotocin-Induced Diabetic Rats

<div><p>Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates several cellular events, including inflammation and tissue remodelling. In this study, we investigated the tissue-specific expression of SPARC in streptozotocin (STZ)-induced diabetes, and found that SPARC was significantly up-regulated in the liver while down-regulated in the pancreas of STZ-induced diabetic rats. Chronic inflammation occurred in the diabetic pancreas accompanied by up-regulation of CCAAT/enhancer-binding protein beta (C/EBPβ) and its targets (TNFα, <i>Il6</i>, CRP, and <i>Fn1</i>) as well as myeloperoxidase (<i>Mpo</i>) and C-X-C chemokine receptor type 2 (<i>Cxcr2</i>). Diabetic liver showed significant up-regulation of <i>Tgfb1</i> as well as moderately less up-regulated TNFα and reduced <i>Fn1</i>, resulting in elevated fibrogenesis. PARP-1 was not up-regulated during CD95-mediated apoptosis, resulting in restoration of high ATP levels in the diabetic liver. On the contrary, CD95-dependent apoptosis was not observed in the diabetic pancreas due to up-regulation of PARP-1 and ATP depletion, resulting in necrosis. The cytoprotective machinery was damaged by pancreatic inflammation, whereas adequate antioxidant capacity indicates low oxidative stress in the diabetic liver. High and low cellular insulin content was found in the diabetic liver and pancreas, respectively. Furthermore, we identified six novel interacting partner proteins of SPARC by co-immunoprecipitation in the diabetic liver and pancreas, and their interactions with SPARC were predicted by bioinformatics tools. Taken together, opposite expression of SPARC in the diabetic liver and pancreas may be related to inflammation and immune cell infiltration, degrees of apoptosis and fibrosis, cytoprotective machinery, and cellular insulin levels.</p></div

Effect of supplementation with powdered persimmon leaf on the glucose regulation in C57BL/KsJ-<i>db/db</i> mice.

<p>A; fasting blood glucose level. B&C plasma insulin level and HOMA-IR. D&E activity and/or mRNA expression of enzymes for hepatic gluconeogenesis and glucose utilization. The mRNA levels were analyzed by Northern blot analysis using GK probes and normalized to an internal control (GAPDH). Three independent analyses were performed. F; hepatic glycogen content. Values are the mean ± S.E., n = 10. *p<0.05, **p<0.01, ***p<0.001. GAPDH; glyceraldehyde-3-phosphate dehydrogenase, G6Pase; glucose-6-phosphatase, GK; glucokinase, HOMA-IR; homeostatic index of insulin resistance, PEPCK; phosphoenolpyruvate carboxykinase, PL; powdered persimmon leaf.</p

Immunohistochemistry of SPARC-interacting partner proteins (CA3, CPS1, RARhoGAP, and BHMT) in liver (A) as well as PA and APC2 in pancreatic tissue (B) from control and diabetic rats.

<p>Representative photomicrographs are shown from sections counter-stained with haematoxylin (magnification X400 and scale bar represents 50 μm).</p

Tissue-dependent protein expression levels of MCP-1, iNOS, NFκB, Cyt C, PARP-1 ATP5B, and CD95 in liver (A and C) and pancreas (B and D), respectively.

<p>Data are representative of three independent experiments. Statistical significance between male and female rats was calculated by One-way ANOVA followed by Tukey’s post hoc tests, where <i>p</i>-value is *<i>p</i><0.05 and **<i>p</i><0.01, and significance between controls and diabetics is represented by ^†<i>p</i><0.05 and ^††<i>p</i><0.01.</p

H&E staining profiles and immunohistochemistry of insulin in liver and pancreas (A), where representative photomicrographs are shown and immunohistochemical sections were counter-stained with haematoxylin (magnification X400 and scale bar represents 50 μm) as well as tissue-dependent protein expression levels of HSP90, SOD2, INS in liver (B) and pancreas (C), respectively.

<p>Data are representative of three independent experiments. Statistical significance between male and female rats was calculated by One-way ANOVA followed by Tukey’s post hoc tests, where <i>p</i>-value is *<i>p</i><0.05 and **<i>p</i><0.01, and significance between controls and diabetics is represented by ^†<i>p</i><0.05 and ^††<i>p</i><0.01.</p

Tissue-dependent protein expression levels of C/EBPβ, TNFα, CRP, and mRNA expression levels of <i>Il6</i>, <i>Fn1</i>, <i>Tgfb1</i>, <i>Mpo</i>, <i>Cxcr2</i>, and <i>Il1b</i> in liver (A and C) and pancreas (B and D), respectively.

<p>Data are representative of three independent experiments. Statistical significance between male and female rats was calculated by One-way ANOVA followed by Tukey’s post hoc tests, where <i>p</i>-value is *<i>p</i><0.05 and **<i>p</i><0.01, and significance between controls and diabetics is represented by ^†<i>p</i><0.05 and ^††<i>p</i><0.01.</p

Effect of supplementation with powdered persimmon leaf on the lipid regulation in C57BL/KsJ-<i>db/db</i> mice.

<p>A; liver weight. B; hepatic triglyceride and cholesterol contents. C; hepatic morphology. Arrows indicate hepatic lipid droplets. H&E staining. Original magnification ×200. D; activity of the enzymes for hepatic fatty acid synthesis and esterification. E; activity of the enzymes for hepatic cholesterol synthesis and esterification. F; activity of the enzymes for hepatic fatty acid oxidation and uptake. G; mRNA expression of lipogenic transcription factor and its target gene. The mRNA levels were analyzed by real-time PCR and normalized to an internal control (GAPDH). H; fecal lipids content. Values are the mean ± S.E., n = 10. *p<0.05, **p<0.01, ***p<0.001. ACAT; acyl CoA: cholesterol acyltransferase, ACL; ATP-citrate lyase, CPT; carnitine palmitoyl transferase, DGAT; glyceraldehyde-3-phosphate dehydrogenase, FAS; fatty acid synthase, HMGR; 3-hydroxy-3-methylglutaryl-coenzyme, PAP; phosphatidate phosphohydrolase, PL; powdered persimmon leaf, PPARγ; peroxisome proliferator activated receptor γ, SCD1; steraroyl-CoA desaturase-1.</p

Representative silver-stained SDS-PAGE images of Co-IP samples in the liver (A) and pancreas (B) for identification of SPARC-interacting partner proteins.

<p>Proteins were identified by MALDI-TOF/MS analysis and are indicated by arrows along with their abbreviated names. Full names of proteins are presented in the abbreviations section. Physical interaction map analyzed by STRING in the liver (C) and pancreas (D). Expression patterns of SPARC-interacting partner proteins identified in the liver (E) and pancreas (F). Data are representative of three independent experiments. Statistical significance between male and female rats was determined by One-way ANOVA followed by Tukey’s post hoc tests, where <i>p-</i>value is *<i>p</i><0.05 and **<i>p</i><0.01. Significance between control and STZ-treated rats is represented by ^†<i>p</i><0.05 and ^††<i>p</i><0.01.</p

Sequences of primers used for quantitative real-time PCR in this study.

<p>Sequences of primers used for quantitative real-time PCR in this study.</p