Metabolic labeling unveils alterations in the turnover of HDL-associated proteins during diabetes progression in mice

Abstract

Several aspects of diabetes pathophysiology and complications result from hyperglycemia-induced alterations in the structure and function of plasma proteins. Furthermore, insulin has a significant influence on protein metabolism by affecting both the synthesis and degradation of proteins in various tissues. To understand the role of progressive hyperglycemia on plasma proteins, in this study, we measured the turnover rates of high-density lipoprotein (HDL)-associated proteins in control (chow diet), prediabetic [a high-fat diet (HFD) for 8 wk] or diabetic [HFD for 8 wk with low-dose streptozotocin (HFD + STZ) in weeks 5-8 of HFD] C57BL/6J mice using heavy water (²H₂O)-based metabolic labeling approach. Compared with control mice, HFD and HFD + STZ mice showed elevations of fasting plasma glucose levels in the prediabetic and diabetic range, respectively. Furthermore, the HFD and HFD + STZ mice showed increased hepatic triglyceride (TG) levels, total plasma cholesterol, and plasma TGs. The kinetics of 40 proteins were quantified using the proteome dynamics method, which revealed an increase in the fractional synthesis rate (FSR) of HDL-associated proteins in the prediabetic mice compared with control mice, and a decrease in FSR in the diabetic mice. The pathway analysis revealed that proteins with altered turnover rates were involved in acute-phase response, lipid metabolism, and coagulation. In conclusion, prediabetes and diabetes have distinct effects on the turnover rates of HDL proteins. These findings suggest that an early dysregulation of the HDL proteome dynamics can provide mechanistic insights into the changes in protein levels in these conditions.NEW & NOTEWORTHY This study is the first to examine the role of gradual hyperglycemia during diabetes disease progression on HDL-associated protein dynamics in the prediabetes and diabetic mice. Our results show that the fractional synthesis rate of HDL-associated proteins increased in the prediabetic mice whereas it decreased in the diabetic mice compared with control mice. These kinetic changes can help to elucidate the mechanism of altered protein levels and HDL dysfunction during diabetes disease progression.

Keywords: HDL; heavy water; mass spectrometry; proteomics; type 2 diabetes.

Graphical abstract

Figure 1.

Experimental design and the workflow of the study. Mice fed either a chow diet (n = 6 mice) or a high-fat diet (HFD; n = 10 mice). After 4 wk, HFD-fed mice were randomized into two groups. The first group continued their HFD for an additional 4 wk to model prediabetes (n = 4/group). The second group of mice on HFD was treated weekly with a low dose of streptozotocin (HFD+STZ) to mimic type 2 diabetes in humans (n = 6/group). Prediabetic, diabetic, and control mice (control) received 30 μL/g bolus dose of ²H₂O followed by 8% ²H₂O in their drinking water during the last 7 days of the study. Blood samples were drawn at different time points during 1 wk of the study. HDL proteins were isolated and analyzed by LC-MS/MS after delipidation and trypsin-digestion. High-resolution mass spectra were collected and processed to 1) identify proteins and their posttranslational modifications and 2) assess protein fractional synthesis rates (k) based on ²H-incorporation.

Figure 2.

Glucose and insulin tolerance tests. A and B: the time-course and the area under the curve (AUC) of the glucose tolerance test (GTT) in the mice after intraperitoneal injection of glucose following an 8-h fast. C and D: the time-course and the area under the curve (AUC) of the insulin tolerance test (ITT) in nonfasted mice after intraperitoneal injection of insulin (0.75 units/kg). Data are presented as means ± SD.

Figure 3.

The effect of high-fat diet (HFD) alone or in combination with streptozotocin (HFD+STZ) on the fractional synthesis of the selected HDL proteins assessed with the ²H₂0 metabolic-labeling technique. The newly made fractions of APOAI (A) and KNG1 (B) were calculated based on ²H incorporation into the peptides TQVQSVIDK and DIPVDSPEK, respectively. The fractional synthesis rate is denoted by the rate constant k. Data are presented as means ± SD. Statistical significance, *P < 0.05 compared with HFD.

Figure 4.

Volcano plot summarizing prediabetes and diabetes-induced changes in HDL proteins turnover. A: prediabetes (HFD) vs. control mice. B: diabetes (HFD+STZ) vs. prediabetes (HFD). C: diabetes vs. control. Red dots represent proteins that changed significantly in turnover rate (adjusted P value < 0.05). Each dot represents a protein. HFD, high-fat diet; STZ, streptozotocin. Full protein names and accession numbers are provided in Supplementary Table S3.

Figure 5.

Comparison of proteome dynamics in control, HFD, and HFD+STZ groups. A: the average turnover rates for all proteins identified in each animal of the control, HFD, and HFD+STZ groups. Data are presented as means ± SD. The mean values indicate the average of total protein turnover for each group (Control: n = 6, HFD: n = 4, and HFD+STZ: n = 6). The P value indicating significant differences is based on each animal as biological replicate. B: the correlation between the turnover rates observed for proteins identified in the HFD and HFD+STZ groups. The equation for the regression curve fitting is presented and indicates a >50% decrease in the turnover of proteins in the HFD+STZ group than the HFD group. HFD, high-fat diet; STZ, streptozotocin.

Figure 6.

Diabetes progression affects HDL proteome dynamics. A: relationships between the turnover rates for proteins overrepresented (FDR < 0.05) compared with the mouse plasma proteome, with functionally related gene ontology (GO) categories in control, prediabetic (HFD), and diabetic (HFD+STZ) mice. Proteins within a pathway exhibit coordinated increased and decreased protein turnover in prediabetic and diabetic states, respectively. Differences in turnover rates are shown for the proteins from the top 3 GO Biological Process (BP) categories that experienced significant changes between at least one of the experimental conditions (ANOVA tests, FDR-corrected P < 0.05). The list of individual proteins in each of these and other GOBP categories is available in the Supplemental Table S6. ANOVA results are shown for each GO BP category. *,#P < 0.05 vs. Control and HFD, respectively. B–D: differences in the turnover rates between the control, high-fat diet (HFD) and the high-fat diet and streptozotocin (HFD+STZ) groups for proteins belong to the acute-phase response (B), coagulation (C), and reverse cholesterol transport pathways (D). *,#P < 0.05 vs. Control and HFD, respectively. ANT3, antithrombin III; APOH, β-2-glycoprotein 1; APOA1, apolipoprotein A-1; APOA2, apolipoprotein A-II; APOA4, apolipoprotein A-IV; ALBU, albumin; CERU, ceruloplasmin; CFAI, complement factor I; CO3, complement C3; FDR, false discovery rate; FIBA, fibrinogen α-chain; HRG, histidine rich glycoprotein; KNG1, Kininogen-1; KLKB1, plasma kallikrein; PLMN, plasminogen; STZ, streptozotocin; THRB, prothrombin; TRFE, serotransferrin; VTDB, vitamin D binding protein.

Figure 7.

The effect of hyperglycemia-induced glycation on albumin turnover. Slow ²H-incorporation into glycated albumin peptide k^GluQTALAELVK at Lys-600 suggests that glycation of albumin inhibits its degradation and contributes to oligomerization.