Our study here showed that ZDF rats (fa/fa) and their basic diet-fed littermate wild-type rats (fa/+) exhibited different plasma proteins and lipids profiles which could distinguish the diabetic status of rats clearly by the hierarchical clustering of DEPs/DELs. GO function enrichment analysis demonstrated that DEPs were in the extracellular, which gave these proteins the potential to become plasma biomarkers. Furtherly, KEGG pathway enrichment analysis of DEPs and DELs revealed the related mechanisms of T2DM, such as oxidative stress, insulin resistance and metabolic disorders. This was consistent with previous researches [31–33]. Some differentially expressed indicators and their role in KEEG pathways led us to believe that they had the potential to be biomarkers, as follows: Down-regulated ceruloplasmin, extracellular superoxide dismutase [Cu-Zn] and glutathione peroxidase 6 indicated a decrease in antioxidant level [34, 35]. Up-regulated glycogen phosphorylase (liver form), AKT serine/threonine kinase 2, 60 kDa heat shock protein (mitochondrial), and down-regulated insulin-like growth factor 1 (isoform CRA_b) proved a significant insulin resistance [36–38]. Up-regulated glyceraldehyde-3-phosphate dehydrogenase, uncharacterized protein and 4-trimethylaminobutyraldehyde dehydrogenase showed an increasing degree of plasma glycolysis [39]. Up-regulated apolipoprotein C-I and apolipoprotein C-II illustrated blood low-density lipoproteins accumulated in the blood, thereby increasing the risk of cardiovascular complications in diabetes [40, 41].
Importantly, we found two interesting points in these screened biomarkers.
Firstly, three oxidative stress-related markers that we screened, ceruloplasmin, extracellular superoxide dismutase [Cu-Zn] and glutathione peroxidase 6, are all trace elements related-metalloproteins. Ceruloplasmin stores approximately 95% of copper in the blood in a non-diffused state [42] and is linked to iron metabolism [43]. More than half of the patients with aceruloplasminemia (ACP), an autosomal recessive genetic disease caused by mutations in the gene encoding ceruloplasmin, have diabetes as their earliest symptom [44]. And some epidemiological studies use ceruloplasmin to indicate diabetes nephropathy progresses [45–47]. Each subunit of extracellular superoxide dismutase [Cu-Zn] contains a copper ion and a zinc ion, and each of the four subunits of glutathione peroxidase 6 contains a single selenium ion. These metal trace elements play a major part in maintaining the normal function of these proteins [48–50]. So, our study provides evidence for the association of T2DM with trace elements, such as copper, zinc, iron, selenium, etc., through metalloproteins.
The second point is a question of lipid metabolism and transport. A significant module of the DEPs PPI network, which contains three up-regulated proteins, apolipoprotein C-I, apolipoprotein C-II (Predicted) and apolipoprotein C-IV, and two down-regulated proteins, apolipoprotein M and very-low-density lipoprotein receptor, suggests metabolism and transport disorder of lipid. Hierarchical clustering of DELs proves this. Since plasma lipids are greatly influenced by diet, we use the basic diet to feed ZDF rats for one week and all animals are fasted for 12 hours before collecting plasma samples. And because of this, we don’t screen biomarkers in DELs. It is noteworthy that our results show the association between the apolipoprotein C and T2DM. Since there are limited studies in this area [51, 52], we will pay more attention to the changes in apolipoprotein C during the progress of T2DM in the future.
The pathogenesis of T2DM is complicated. Multi-omics study helps to profoundly understand the molecular mechanisms and explores the possible directions in diagnosis and treatment of it. Screening of plasma biomarkers has unparalleled advantages, as the plasma is more stable and more readily available compared to urine and tissues, respectively [53, 54]. We screened the potential biomarkers of T2DM by comparing the plasma proteins and lipids expression levels in ZDF rats (fa/fa) and their basic diet-fed littermate wild-type controls (fa/+). The comparison method we adopted fully considered the influence of genetics and environments. Although this comparison will overestimate the role of the genetic effects of the leptin receptor gene in T2DM and increase the difficulty of comparison with other similar studies [55, 56], we believe this is a simple and effective comparison strategy when the population's genetic background is not known clearly. So far, very limited studies have been performed with regard to detection of plasma proteins and lipids profiles in ZDF rats. Therefore, this study may provide a novel strategy to characterize the molecular mechanism of T2DM and search for potential biomarkers [52, 57, 58], despite the fact that this is only at the animal level. It is notable that the samples number is small, although this is sufficient for LC-MS/MS analysis. Increasing samples and verifying the predictability of these candidate biomarkers are the focus of our next work.