Effect of High-Fat Diet on Hepatic Proteomics of Hamsters

A high-fat diet contributes to the etiology of metabolic diseases. As the liver plays a crucial role in metabolism, an insight into the hepatic proteomics will help to illustrate the physiological effect of a high-fat diet. Fourteen nine-week old male Syrian hamsters were maintained on either control (C) or high-fat (HF) diets (0.2% cholesterol +22% fat) for 8 weeks. Hamsters were chosen because they show close similarity to human lipid metabolism. At the end of study, blood and livers were collected for analysis. Liver proteins were fractionated by electrophoresis, digested by trypsin, and then separated by label-free nano-LC/MS/MS. The TurboSequest algorithm was used to identify the peptide sequences against the hamster database in Universal Proteins Resource Knowledgebase (UniProt). The results indicate that 1191 hepatic proteins were identified and 135 of them were expressed differentially in the high-fat group (<i>p</i> < 0.05). Some of these 135 proteins that involve in metabolic diseases were further validated by Western blotting. The animals maintained on the high-fat diet had significantly (<i>p</i> < 0.05) higher serum triglyceride, cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and uric acid. Animals consuming a high-fat diet also had significantly (<i>p</i> < 0.05) more accumulation of triglyceride and cholesterol in livers. Xanthine dehydrogenase (XDH), which plays an important role in uric acid synthesis, was up-regulated by the high-fat diet (<i>p</i> < 0.05). The α-subunit of hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (HADHA), which catalyzes the second and third reactions of β-oxidation, was down-regulated by the high-fat diet (<i>p</i> < 0.05). Aconitate hydratase 2 (ACO2), which catalyzes the conversion of citrate to isocitrate in TCA cycle, was down-regulated in animals of the high-fat group (<i>p</i> < 0.05). Inflammatory markers annexin A3 (ANXA3) and annexin A5 (ANXA5) were up-regulated by the high-fat diet (<i>p</i> < 0.05). Moreover, enzymes involved in the urea cycle were suppressed by high-fat diet, including carbamoyl phosphate synthase 1 (CPS1), ornithine transcarbamoylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), and arginase 1 (ARG 1). Post-translational modifications (PTM) of ANXA3, ANXA5, and XDH were also analyzed. A set of differentially expressed proteins were identified as molecular markers for elucidating the pathological mechanism of high-fat diet

Consumption of Purple Sweet Potato Affects Post-Translational Modification of Plasma Proteins in Hamsters

A high level of intake of vegetables is strongly associated with the prevention of chronic diseases. Because post-translational modifications (PTMs) have been shown to be the important biomarkers of the change in physiological functions, this study aimed to explore the changes in PTMs of plasma proteins when purple sweet potato (PSP), a root vegetable, was incorporated into the daily diet. Male Syrian hamsters were maintained on a rice diet (50% rice) or PSP diet (25% rice and 25% PSP) for 12 weeks. Plasma proteins were fractionated by electrophoresis, digested by trypsin, and then separated by nano-liquid chromatography and tandem mass spectrometry. The TurboSequest algorithm was used to identify peptide sequence against the hamster database in Universal Proteins Resource Knowledgebase, and in-house PTM finder programs were used for identification and quantification of PTMs. The results indicated that 95 plasma proteins were identified and 28 PTM sites on 26 of these 95 proteins were affected by consumption of PSP (p < 0.05). Methylation accounted for the largest percentage of affected modifications (35.71%). This study also showed that incorporation of purple sweet potato into the diet significantly lowered blood and liver lipids (p < 0.05). The results of this study provide a basis for prospective studies evaluating the effects of dietary intervention on modifications of proteins

Cox proportional hazard model for prognosis of HD patients at a four-year follow-up.

a<p>Values are expressed as mean ± SD or percent.</p>b<p>ACE, angiotensin converting enzyme; ARB, angiotensin receptor blocker.</p>c<p>CI, confidence interval.</p>d<p>HR, hazard ratio.</p>*<p><i>p</i><0.10 in univariate Cox regression analysis.</p

Box plots of plasma vitamin D binding protein and clusterin levels in HD patients.

<p>ELISA validation of vitamin D binding protein and clusterin concentration for patients’ plasma originally analyzed by 2-DE. Long-term HD survivors have (a) higher vitamin D binding protein (204.5±63.9 mg/L vs. 149.0±71.6 mg/L, p = 0.036) and (b) lower clusterin (282.3±105.8 mg/L vs. 438.5±174.2 mg/L, p = 0.011) than the short-term HD patients, which are concordant with 2-DE expression pattern.</p

Consumption of Purple Sweet Potato Affects Post-Translational Modification of Plasma Proteins in Hamsters

A high level of intake of vegetables is strongly associated with the prevention of chronic diseases. Because post-translational modifications (PTMs) have been shown to be the important biomarkers of the change in physiological functions, this study aimed to explore the changes in PTMs of plasma proteins when purple sweet potato (PSP), a root vegetable, was incorporated into the daily diet. Male Syrian hamsters were maintained on a rice diet (50% rice) or PSP diet (25% rice and 25% PSP) for 12 weeks. Plasma proteins were fractionated by electrophoresis, digested by trypsin, and then separated by nano-liquid chromatography and tandem mass spectrometry. The TurboSequest algorithm was used to identify peptide sequence against the hamster database in Universal Proteins Resource Knowledgebase, and in-house PTM finder programs were used for identification and quantification of PTMs. The results indicated that 95 plasma proteins were identified and 28 PTM sites on 26 of these 95 proteins were affected by consumption of PSP (<i>p</i> < 0.05). Methylation accounted for the largest percentage of affected modifications (35.71%). This study also showed that incorporation of purple sweet potato into the diet significantly lowered blood and liver lipids (<i>p</i> < 0.05). The results of this study provide a basis for prospective studies evaluating the effects of dietary intervention on modifications of proteins

Kaplan-Meier analysis of plasma DBP level in HD patients.

<p>HD patients with the lowest plasma vitamin D binding protein level were at the highest risk for mortality than those with the top tertile plasma levels (p = 0.03; log-rank test).</p

Representative gel sections of protein alterations related to HD duration.

<p>Each bar represents the intensity of the squared spot quantitatively analyzed by PDQuest software. Circles were used to indicate the α1-antitrypsin and fibrinogen γ chain respectively in (h) and (i) according to SWISS-2DPAGE (<a href="http://tw.expasy.org/ch2d/" target="_blank">http://tw.expasy.org/ch2d/</a>).</p

Demographics and biochemistry of the studied HD population.

<p>Demographics and biochemistry of the studied HD population.</p

Plasma Protein Characteristics of Long-Term Hemodialysis Survivors

<div><p>Hemodialysis (HD) patients are under recurrent circulatory stress, and hemodialysis has a high mortality rate. The characteristics of plasma proteomes in patients surviving long-term HD remain obscure, as well as the potential biomarkers in predicting prognoses. This study reports the proteome analyses of patient plasma from non-diabetic long-term HD (LHD, dialysis vintage 14.9±4.1 years, n = 6) and the age/sex/uremic etiology-comparable short-term HD (SHD, dialysis vintage 5.3±2.9 years, n = 6) using 2-DE and mass spectrometry. In addition, a 4-year longitudinal follow-up of 60 non-diabetic HD patients was subsequently conducted to analyze the baseline plasma proteins by ELISA in predicting prognosis. Compared to the SHD, the LHD survivors had increased plasma vitamin D binding proteins (DBP) and decreased clusterin, apolipoprotein A-IV, haptoglobin, hemopexin, complement factors B and H, and altered isoforms of α1-antitrypsin and fibrinogen gamma. During the 45.7±15 months for follow-up of the 60 HD patient cases, 16 patients died. Kaplan-Meier analysis demonstrated that HD patients with the lowest tertile of the baseline plasma DBP level have a significantly higher mortality rate. Multivariate Cox regression analysis further indicated that DBP is an independent predictor of mortality. In summary, the altered plasma proteins in LHD implicated accelerated atherosclerosis, defective antioxidative activity, increased inflammation/infection, and organ dysfunction. Furthermore, lower baseline plasma DBP in HD patients is related to mortality. The results suggest that the proteomic approach could help discover the potential biomarker in HD prognoses.</p> </div

Lists of differentially expressed proteins in the plasma of long-term HD compared to the short-term HD patients.

a<p>Protein identification was done by the peptide mass fingerprinting (pmf) using MALDI-TOF MS, or by sequencing amino acids using Q-TOF tandem MS, as shown in the supplementary materials including <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040232#pone.0040232.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040232#pone.0040232.s002" target="_blank">Data S1</a>.</p