Label-Free Proteomics of the Fetal Pancreas Identifies Deficits in the Peroxisome in Rats with Intrauterine Growth Restriction

Abstract

Aim: The objective of the present study was to identify differentially expressed proteins (DEPs) in the pancreas of a fetus with intrauterine growth restriction (IUGR) and to investigate the molecular mechanisms leading to adulthood diabetes in IUGR.

Methods: The IUGR rat model was induced by maternal protein malnutrition. The fetal pancreas was collected at embryonic day 20 (E20). Protein was extracted, pooled, and subjected to label-free quantitative proteomic analysis. Bioinformatics analysis (GO and IPA) was performed to define the pathways and networks associated with DEPs. LC-MS results were confirmed by western blotting and/or quantitative PCR (q-PCR). The principal parameters of oxidative stress-superoxide dismutase (Sod) were determined in blood samples of fetal rats.

Results: A total of 57 DEPs (27 upregulated, 30 downregulated) were identified with a 1.5-fold change threshold and a p value ≤ 0.05 between the IUGR and the control pancreas. Bioinformatics analysis revealed that these proteins play important roles in peroxisome biogenesis and fission, fatty acid beta-oxidation (FAO), mitotic cell cycle, and histone modification. The peroxin Pex14 was downregulated in the IUGR pancreas as confirmed by western blotting and q-PCR. Pmp70, a peroxisomal membrane protein involved in the transport of fatty acids, was upregulated. Hsd17b4 and Acox1/2, which catalyze different steps of peroxisomal FAO, were dysregulated. Sod plasma concentrations in the IUGR fetus were higher than those in the control, suggesting partial compensation for oxidative stress. Multiple DEPs were related to the regulation of the cell cycle, including reduced Cdk1, Mcm2, and Brd4. The histone acetylation regulators Hdac1/2 were downregulated, whereas Sirt1/3 and acetylated H3K56 were increased in the IUGR fetal pancreas.

Conclusion: The present study identified DEPs in the fetal pancreas of IUGR rats by proteomic analysis. Downregulation of pancreas peroxins and dysregulation of enzymes involved in peroxisomal FAO may impair the biogenesis and function of the peroxisome and may underlie the development of T2 diabetes mellitus in adult IUGR rats. Disorders of cell cycle regulators may induce cell division arrest and lead to smaller islets. The present data provide new insight into the role of the peroxisome in the development of the pancreas and may be valuable in furthering our understanding of the pathogenesis of IUGR-induced diabetes.

Figure 1

Outline of the experimental workflow.

Figure 2

Global protein expression patterns in the rat fetal pancreas. (a) Venn diagram showing 1,497 proteins identified in both groups (overlap), 36 proteins detected only in the control group (blue), and 64 proteins detected only in the IUGR group (yellow). (b) Principal component analysis (PCA) graph showing a clear separation between control and IUGR samples. Each point in the PCA graph represents the protein profile of one biological replicate sample. (c) K-means clustering representation of the profiles of 57 differentially expressed proteins. The percentage variation is represented by a color scale (top right) from low (blue) to high (red). (d) Volcano plots of the 1,497 quantified proteins showing the distribution of significance and fold change of identified proteins (the logarithmic ratio of protein LFQ intensities in the IUGR/CON comparison was plotted against negative logarithmic p values of the t-test). Vertical dotted lines mark a fold change of ±150%, and horizontal dotted lines mark a p < 0.05.

Figure 3

GO annotation and functional enrichment of differential proteins (including 57 expressed proteins and 100 no/have proteins).GO terms for subcellular location distribution (a), molecular functions (b), and biological process (c). (d) Enrichment analysis shows the top 20 enriched GO terms associated with differential proteins.

Figure 4

Ingenuity pathway analysis identified the top functions and canonical pathways associated with differential proteins. (a) Top 10 diseases and functions associated with DEPs in the fetal pancreas. (b) Top10 canonical pathways associated with DEPs in the fetal pancreas. Red, green, and gray indicate the percent of upregulated, downregulated, or no change proteins that matched each pathway, respectively. The orange line indicates the p value of the association between the reference and focus proteins for each pathway. The number on top of each pathway indicates the total number of proteins associated with the corresponding pathway.

Figure 5

IPA identified overlapping canonical pathways and predicated networks associated with differential proteins. (a) Overlapping canonical pathways associated with differential proteins. (b) Predicted protein interaction network was generated with differential proteins associated with peroxisome and cell cycle by IPA.

Figure 6

Box plot for the LFQ intensity of focus DEPs associated with the peroxisome and cell cycle in LFQ proteomic analysis. (a) Peroxisome-related factors. (b) Cell cycle regulators. (c) HDAC1. (d) Label-free analysis of focus proteins detected in control or IUGR specimens. The results were expressed as the mean ± SEM. n = 3 per group, ^∗p < 0.05 vs. control.

Figure 7

Investigation of peroxisomal-related proteins in the fetus. (a) Western blot analysis of peroxisome-related factors in the fetal pancreas. (b) Densitometry analysis results were expressed as the mean ± SEM. n = 5–8, ^∗p < 0.05 vs. control. (c) The mRNA levels of focus proteins in the fetal pancreas were determined by quantitative RT-PCR with β-actin as the reference gene. (d) Representative photomicrographs of immunofluorescence analysis of Pex14 in sections of the pancreas from the control (A, B, C) and IUGR (D, E, F) (original magnification 400x). (e) The STRING network of peroxins and peroxisomal FAO with altered expression. Colored lines between the proteins indicate the various types of interaction evidence. (f) Schematic representation of the peroxisomal FAO, which consists of 4 steps catalyzed by different enzymes and leading to the formation of a chain-shortened acyl-CoA and acetyl-CoA.

Figure 8

Peroxisomal-related proteins in adult offspring. (a) Western blot analysis of critical peroxisome factors in the adult pancreas. (b) Densitometry analysis results were expressed as the mean ± SEM. n = 6–8, ^∗p < 0.05 vs. age-matched control.

Figure 9

Cell cycle regulators and Hdacs. (a, b) Representative immunoblotting and densitometric quantification of cell cycle regulators in the fetal pancreas. (c, d) Representative immunoblotting and densitometry analysis of Hdacs in the fetal pancreas. Results were expressed as the mean ± SEM. n = 6–8, ^∗p < 0.05 vs. control. (e) The mRNA levels of these proteins in the fetal pancreas were determined by quantitative RT-PCR with β-actin as the reference gene (n = 6 per group, ^∗p < 0.05, vs. control). (f) The STRING networks of cell cycle regulators and Hdacs with altered expression. Colored lines between the proteins indicate the various types of interaction evidence.