Introduction
Patients with type 1 diabetes (T1DM) are at increased risk for cardiovascular disease [
1]. This risk starts early in life as there is evidence of subclinical cardiovascular disease (CVD) in youth with T1DM [
2]. The primary risk factors that have been attributed to CVD in this population include poor glycemic control, hypertension, obesity, and dyslipidemia [
1,
3]. Dyslipidemia, characterized by non-HDL cholesterol > 130 mg/dL was found in 27.7% and low HDL < 35 mg/dL in only 3.4% of a large cohort of 682 children with T1DM [
4]. In the SEARCH study, among 512 youth with T1DM and 188 healthy controls, the prevalence of low HDL < 35 mg/dL was 10.3%, 7.6% and 5% among healthy children and youth with T1DM and optimal or suboptimal control, respectively [
5]. In general, low HDL levels in patients with T1DM is not as frequent as other types of dyslipidemia and the HDL profile can often even be favorable in patients with T1DM. However, despite the normal HDL cholesterol, the function of HDL can be impaired in some patients with T1DM [
6‐
8]. The primary cardioprotective functions of HDL include the following: (i) prevention of the oxidation of LDL (ii) cholesterol efflux from the vessel wall and transport of cholesterol to the liver and (iii) anti-inflammatory function [
9].
Alterations in the protein composition of HDL can affect its protective functions [
7,
10]. For example, adults with T1DM and low levels of the protein apolipoprotein A-I (apoA-I) on the HDL particles were more likely to develop CVD [
7]. Adult patients with T1DM and subclinical atherosclerosis have decreased levels of the potent antioxidant protein paraoxonase-3 (PON3) and PON3 concentration correlates with the anti-inflammatory function of HDL [
9]. HDL proteome studies done in patients with type 2 diabetes (T2DM) have found alterations of HDL proteins linked to increased CVD risk [
11,
12].
The influence of T1DM on the HDL proteome in youth with T1DM has not been reported. Our goal was to test the hypothesis that youth with T1DM have protein alterations on HDL that could play a role in modifying HDL function and contribute to an increased risk for CVD. In this study, SWATH mass spectrometry was used for label-free relative quantification of the HDL proteome. The protein composition of HDL was compared between youth with T1DM and healthy controls (HC) and the effect of glycemic control on HDL bound proteins was evaluated.
Discussion
This report describes, for the first time, proteomic alterations of HDL in a cohort of young adults with T1DM. We have identified several HDL-bound proteins that are significantly affected in patients with T1DM. We observed three clear trends in those affected proteins. FHR2 was increased in T1DM and not corrected by glucose control. This may suggest that this is caused by some consequence of the underlying condition and not circulating glucose levels. A1BG and ITIH4 were increased in T1DM and partially corrected by glucose control. Finally, CO3 and ALB are decreased in T1DM patients with well-controlled glucose. This may suggest the administration of insulin is influencing the association of these proteins with HDL.
Gene ontology analysis using PANTHER protein classifications revealed a significant enrichment of proteins with protease inhibitor function, particularly serine protease inhibitors. This indicates that the effect of T1DM on the HDL proteome may specifically influence the protease regulator activity of HDL. Protease regulator activity on HDL has been previously suggested to play an important role in atherosclerotic cardiovascular disease [
24]. The following paragraphs discuss some potential roles for each of the affected proteins in HDL function and CVD risk.
FHR2, is a complement factor H (CFAH) related protein that resembles structurally and immunologically the complement factor H. While the complement pathway is a critical component of innate immunity, inappropriate complement activation has been linked to inflammation, diabetes, insulin resistance, atherosclerosis, and cardiometabolic diseases [
25,
26]. Complement factor H is an important regulatory protein and prevents tissue damage from inappropriate complement activation [
27]. Recently, it was shown that factor H binds to ApoE on HDL and this plays a role in regulating complement activation [
28]. Furthermore, CFAH presence has also been linked with decreased insulin production from rat pancreatic cells and it may play a role in the pathogenesis of diabetes [
27]. Whether the increased FHR2 in HDL of T1DM patients is related to the pathogenesis of T1DM remains to be explored. FHR2 has been previously detected on lipoproteins and it is thought to facilitate the adhesive response of neutrophils to lipopolysaccharides [
27]. It is also possible that complement factor H and complement factor H related proteins (such as FHR2) play a role in lipid transport and regulate lipid homeostasis [
29]. The mechanism by which CFAH and FHR2 interact to regulate HDL function in diabetes requires further investigation.
A1BG, alpha-1-B glycoprotein, belongs to the immunoglobulin family and its function is largely unknown [
30,
31]. A1BG was found to be overexpressed in tissues of pancreatic ductal adenocarcinoma and liver cancer cell lines [
30], while it was not detected in normal pancreatic tissue and hence could be useful as a tumor marker. A1BG was also reported to be elevated in the urine of normoalbuminuric patients with T1DM when compared to healthy controls; however, the urine levels of this protein did not correlate with HbA1c or urine microalbumin [
31]. Whether this protein could serve as a urine biomarker of early diabetic kidney disease remains to be examined. Interestingly, pharmacogenomic studies have shown that polymorphisms in the
A1BG gene can play a role in cardiovascular outcomes of patients treated with antihypertensive medications [
32].
ITIH4, inter-alpha-trypsin inhibitor heavy chain H4, belongs to the liver-restricted serine protease inhibitor family. It is highly expressed in liver development and low levels have been found in hepatocellular and ovarian cancer [
33,
34]. ITIH4 is elevated in urine of patients with T2DM and microalbuminuria and could possibly serve as a biomarker for diabetic kidney disease [
35]. ITIH4 was also found to be downregulated after a very low caloric diet in patients with T2DM, possibly representing a biomarker of metabolic improvement [
36].
We were unable to identify a clinical basis for the bimodal distribution of ITIH4 in the poorly controlled T1DM cohort. Using logistic regression analysis, high vs low ITIH4 grouping does not appear to correlate with age, sex, BMI, hsCRP, or duration of diabetes. However, the high ITIH4 group, which made up 66% of the poorly controlled cohort displayed a robust elevation compared to HC that, similar to A1BG, was partially corrected with glycemic control. These results suggest that better glycemic control could reverse the changes associated with overexpression of A1BG and ITIH4 on HDL. The fact that some protein alterations can be corrected by glycemic control and others cannot indicates that these HDL proteome effects may be mediated by different mechanistic pathways. Future studies could examine how glycemic control alters HDL structure and function and explore novel mechanisms to improve CVD risk in T1DM.
ALBU, albumin, is the most abundant protein in the plasma and binds to electrolytes, hormones, fatty acids and drugs. Relatively low serum albumin has also been used as a marker of increased mortality from CVD [
37,
38]. Serum albumin has been found to be positively correlated with HDL and total cholesterol, and it is possible that low serum albumin reflects abnormalities in lipid metabolism and function [
39]. Interestingly, glycated albumin was shown to decrease the anti-inflammatory function of HDL and impair the reversed cholesterol transport function, contributing to the development of CVD in patients with diabetes [
40]. Whether the low albumin on HDL we found in our patients with T1DM reflects an imbalance between glycosylated and non-glycosylated forms of albumin requires further study.
CO3, complement factor 3, is produced by macrophages and plays a key initiating role in the activation of complement on the vascular endothelium, which triggers an inflammatory response, creating a vessel wall that is prone to atherosclerosis and increasing CVD risk [
26]. CO3 has been previously detected on HDL [
10,
41]. Vaisar et al. reported multiple complement regulatory proteins on HDL fractions [
41]. Interestingly, in their study, subjects with cardiovascular disease had significantly elevated levels of CO3 on HDL fractions [
41]. Others have shown that increased CO3 on HDL of patients with CVD, psoriasis and rheumatoid arthritis is linked to decreased cholesterol efflux [
10,
42,
43]. Additionally, HDL-bound CO3 has been correlated with increased non-calcified plaque burden in patients undergoing coronary CT angiography [
44]. The current study found lower CO3 in T1DM compared to HC (although statistically significant only in the “well-controlled” glycemic cohort), which suggests a protective effect of insulin given the evidence in literature. This could represent a protective role of HDL against CVD in T1DM subjects during the early stages of disease.
Both ALBU and CO3 were significantly lower only in the group of T1DM with optimal control compared to HC, suggesting that perhaps higher doses of insulin may play a role in altering their values. Insulin can increase the transcapillary escape of albumin as well as the urinary excretion of albumin and whether the lower albumin and CO3 on HDL could be secondary to these mechanisms requires further investigation [
45,
46].
Strengths of our study include that this is a well-characterized cohort of youth with T1DM, with optimal and suboptimal glycemic control, along with detailed HDL proteome data. The combination of two-step size-exclusion chromatography and lipid interaction based HDL purification and SWATH-MS provides sensitive and robust label-free proteomic quantitation for multiple clinical samples. Limitations include the small sample size and lack of full functional studies for each protein of interest. Our goal for this project was to provide an initial characterization of the proteomic differences in HDL composition between youth with T1DM and HC and identify associations with glycemic control. Future studies will further investigate the functional roles of the identified proteins that differed on HDL from T1DM patients.
Our group has previously published differences in the cholesterol efflux values between T1DM and HC in a larger cohort [
8], but in this particular subset of participants we did not detect differences in cholesterol efflux, potentially because of the smaller sample size, the well-controlled status in half of the T1DM participants and their short diabetes duration. There were no significant correlations between the specific proteins of interest and cholesterol efflux. The lack of a group wide effect on HDL’s cholesterol efflux capacity, is not necessarily surprising considering that the HDL-cholesterol levels are not different in this cohort and the protein changes detected here do not include proteins known to influence efflux (e.g. apoA-I, serum amyloid A, etc.) [
44,
47]. Based on our functional classification analysis, it seems likely the influence of T1DM on HDL may be more strongly tied to HDL’s roles in protease regulation and inflammation [
24].
Authors’ contributions
EG is the Principal Investigator of the clinical study, collected and analyzed the data and was a major contributor in writing the manuscript. JM performed the proteomic MS experiments, edited and reviewed the manuscript. MP performed the efflux studies. MP, NM, RG, AR interpreted the patient data and helped in writing the manuscript. SG collected HDL fractions and prepared samples for proteomic experiments, analyzed and interpreted the data and contributed to writing the manuscript. All authors read and approved the final manuscript.