During our analysis, the initial SEREX screening identified PCK1 as an antigen as recognized by serum IgG in patients with atherosclerosis, and subsequently, recombinant GST-tagged PCK1 protein of 301 amino acids was purified. Using recombinant PCK1 protein as an antigen, we examined serum antibody levels using AlphaLISA. The results showed that significantly higher s-PCK1-Ab levels were observed exclusively in patients with DM, but not in those with AIS, TIA, and OSAS, compared with those in HDs (Fig. 1a, 2a,b, Tables 1 − 4). Patients with CVD showed a minimal significant difference from HDs. The AUC value of s-PCK1-Abs versus DM was 0.7024 (Fig. 1b). This DM-specific association of s-PCK1-Ab is distinct from previous results that showed that most of the SEREX autoantibodies screened using sera from patients with atherosclerosis were associated with multiple atherosclerosis-related diseases such as AIS and CVD [13, 14].
The comparison of patients' data with the Mann–Whitney U test and Spearman's correlation analysis showed a close correlation between s-PCK1-Ab levels and hypertension (Tables 4 and 5). Antibody levels did not significantly correlate with blood sugar (BS) (P = 0.4358), HbA1c (P = 0.4515), or glycoalbumin (P = 0.1098), which are typical DM markers (Table 5).
PCK1 is an enzyme that converts oxaloacetic acid to phosphoenolpyruvate; there are two isozymes in humans, mitochondrial (PCK 2) and cytosolic (PCK1) PCK [16–18]. PCK1 is involved in gluconeogenesis and is a crucial enzyme in glucose metabolism in the body [19]. Indeed, PCK1-knockout mice die early after birth with profound hypoglycemia [20], which was partially rescued by the overexpression of PCK1 in the liver [21]. Therefore, gluconeogenesis under the control of PCK1 in the liver is crucial to avoid hypoglycemia. In contrast, the overexpression of PCK1 in mice leads to diabetes [22]. It has also been reported that a − 232C/G containing promoter of PCK1 showed 5- to 100-fold increased basal expression of PCK1 compared with − 232C, and − 232C/G polymorphism of PCK1 has been associated with an increased risk of type 2 DM [23–25]. Glucagon, glucocorticoid, and retinoic acid increased the expression of PCK1, whereas insulin inhibited its expression [26]. Therefore, PCK1 expression increases in the presence of insulin resistance.
Our results showed that s-PCK1-Ab levels were higher in patients with DM than in HDs. Since PCK1 expression seems to increase under diabetic conditions, it might make sense that s-PCK1-Ab levels were higher among patients with DM. One of the major reasons for the development of autoantibodies could be the destruction of lesion tissue, followed by the leakage of intracellular antigenic proteins. Although the leaked proteins may rapidly degrade, repeated leaking and exposure to antigens can tremendously elevate antibody levels. Autoantibody markers are more sensitive than antigen markers. This means that antibody markers can detect very early stages of disease progression [11]. Thus, it is not surprising that antibody markers predict the fate several years later. Since patients with DM, especially those with insulin resistance, often complicate nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, there is a high chance of PCK1 leaking from live cells, which might produce antibodies against PCK1 [27]. Insulin resistance develops not only in atherosclerotic vascular disease but also in hypertension; therefore, s-PCK1-Ab levels are related to CVD and hypertension.
Apart from the complications and comorbidities of DM, it is intriguing that s-PCK1-Ab levels are related to prognosis. Since the mortality rate of DM is much higher than that of HDs, prognostic markers can be used to detect high-risk patients with poor prognosis among patients with DM. Thus, s-PCK1-Ab may be a useful marker. However, we still do not know why high s-PCK1-Ab levels are related to poor prognosis. One reason for this may be the high incidence of CVD. Indeed, it has been reported that a functional promoter polymorphism in PCK1 is associated with carotid wall thickness [28], which is associated with CVD. PCK1 and PCK2 have also been reported to be critical for the growth of certain cancers [29]. Therefore, high levels of s-PCK1-Ab may be related to a high incidence of cancer. The reason for death has not been investigated in all patients with DM who were followed up in this study. Therefore, in future studies, we will identify why high s-PCK1-Ab levels are related to poor prognosis.
This study had some limitations. First, s-PCK1-Ab was measured among the specimens collected at a Japanese university and hospital, which may have biased the specimen population. Second, we do not know the mechanism by which s-PCK1-Abs was increased in patients with DM and hypertension and was related to poor prognosis.