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Home , Apolipoprotein, Apolipoprotein C

1148 Diabetes Care Volume 37, April 2014

Benjamin Bouillet,1,2 Thomas Gautier,2 Glycation of C1 Denis Blache,2 Jean-Paul Pais de Barros,2 Laurence Duvillard,2 Jean-Michel Petit,1,2 Impairs Its CETP Inhibitory Laurent Lagrost,2 and Bruno Verges` 1,2 Property: Pathophysiological Relevance in Patients With Type 1 and Type 2 Diabetes

OBJECTIVE Apolipoprotein (apo)C1 is a potent physiological inhibitor of cholesteryl ester transfer (CETP). ApoC1 operates through its ability to modify the elec- trostatic charge at the surface. We aimed to determine whether the inhibitory ability of apoC1 is still effective in vivo in patients with diabetes and whether in vitro glycation of apoC1 influences its electrostatic charge and its CETP inhibitory effect.

RESEARCH DESIGN AND METHODS ApoC1 concentrations and CETP activity were measured in 70 type 1 diabetic (T1D) patients, 113 patients with type 2 diabetes, and 83 control subjects. The conse- quences of in vitro glycation by methylglyoxal on the electrostatic properties of apoC1 and on its inhibitory effect on CETP activity were studied. An isoelectric analysis of apoC1 was performed in patients with T1D and in normolipidemic- normoglycemic subjects.

RESULTS An independent negative correlation was found between CETP activity and apoC1 1Service Endocrinologie, Diabetologie´ et in control subjects but not in patients with diabetes. HbA1c was independently Maladies Metaboliques,´ University Hospital of

CARDIOVASCULAR AND METABOLIC RISK associated with CETP activity in T1D patients. In vitro glycation of apoC1 modified Dijon, Dijon, France its electrostatic charge and abrogated its ability to inhibit CETP activity in a 2INSERM, UMR 866, University of Bourgogne, concentration-dependent manner. The isoelectric point of apoC1 in T1D patients Dijon, France was significantly lower than that in control subjects. Corresponding author: Benjamin Bouillet, [email protected]. CONCLUSIONS Received 25 June 2013 and accepted 5 December 2013. The ability of apoC1 to inhibit CETP activity is impaired in patients with diabetes. Glycation of apoC1 leads to a change in its electrostatic properties that might Clinical trial reg. no. NCT00953498, clinicaltrials .gov. account, at least in part, for a loss of constitutive CETP inhibition and an increase in © 2014 by the American Diabetes Association. plasma CETP activity in patients with diabetes. See http://creativecommons.org/licenses/by- Diabetes Care 2014;37:1148–1156 | DOI: 10.2337/dc13-1467 nc-nd/3.0/ for details. care.diabetesjournals.org Bouillet and Associates 1149

Apolipoprotein (apo)C1, a small, basic might impair its ability to inhibit CETP serum separation within 2 h after apolipoprotein mainly produced by the activity. We also hypothesized that the collection. The plasma was divided into liver (1), has been shown to play a physicochemical properties of apoC1 might aliquots and stored at 2808C until significant and complex role in VLDL and be altered in diabetes and might result in biological analysis. HDL metabolism. ApoC1 was found to the impairment of its ability to modulate stimulate hepatic production of VLDL CETP activity. Biochemical Assay Fasting plasma glucose, plasma (2), to inhibit the hydrolysis of VLDL In the first part of the current study, the creatinine, fasting total and HDL-C, and (3–5), and to impair the recognition of association between apoC1 TG were measured on a Vista analyzer VLDL by its cellular receptors (6,7). In concentration and CETP activity was with dedicated reagents (Siemens vivo studies in animal models support assessed in patients with type 1 (T1D) Healthcare Diagnostics, Deerfield, IL). the hypothesis that apoC1 plays a and type 2 (T2D) diabetes in comparison LDL-C was calculated by the Friedewald complex and significant role in both the with healthy normolipidemic- equation when TG levels were ,3.87 accumulation of VLDL particles in the normoglycemic individuals. In the mmol/L (20). When TG levels were stream and the reduction in their second part, the effect of in vitro .3.87 mmol/L, LDL-C was measured cholesteryl ester content relative to glycation of apoC1 on its electrostatic directly on the Vista analyzer with (TG) (8–11). charge properties was investigated and dedicated reagents (Siemens Healthcare the consequences of this on its ability to In addition, apoC1 has been shown to be a Diagnostics). HbA was measured by inhibit CETP activity were determined. 1c potent inhibitor of cholesteryl ester high-performance liquid chromatography This study provides evidence of the role transfer protein (CETP) activity. In vitro, on a Variant II device (Bio-Rad, of glycation of apoC1 on the loss of the apoC1 inhibits CETP activity when Richmond, CA) (normal range 4–6% CETP inhibitory effect of apoC1 associated with the plasma HDL fraction [20–60 mmol/mol]). (12). The physiological relevance of apoC1 observed in T1D and T2D patients. as a CETP inhibitor has been shown in ApoC1 Concentration RESEARCH DESIGN AND METHODS apoC1–knocked out/ CETP ApoC1 was measured in total plasma transgenic and human apoC1 transgenic/ Study Design and Patients by a specific ELISA using an anti-human Seventy T1D patients, 113 T2D patients, human CETP transgenic mice (13,14). The apoC1 antiserum from rabbit as and 83 normoglycemic-normolipidemic inhibitory effect of apoC1 was found to rely, previously described (21). control subjects were studied. All at least in part, on its ability to modulate subjects were older than 18 years. Measurement of Cholesteryl Ester the electrostatic charge at the HDL surface, Transfer Activity Using a Radioactivity which is recognized as a major determinant Control subjects were not taking any Method of the CETP-lipoprotein interaction and treatment that could interfere with lipoprotein metabolism (-lowering Cholesteryl ester transfer activity in thus of the exchange of core neutral individual plasma samples (25 mLper (15,16). In healthy, normolipidemic agents, anti-HIV agents, combined oral contraceptive pill, corticoids, or retinoic assay) was the measured transfer subjects, the concentration of plasma from [3H]cholesteryl ester–containing apoC1 has been shown to correlate acid), none were pregnant, the fasting , HDL (2.5 nmol per assay) toward negatively with CETP activity and positively glycemia level was 6.1 mmol/L, the TG level was ,1.7 mmol/L, and the LDL endogenous apoB-containing with plasma HDL (HDL-C) levels 3 , . [ H]cholesteryl ester– (17,18). In contrast, such relationships cholesterol (LDL-C) level was 4.128 mmol/L. containing HDL was obtained with were not observed in nondiabetic patients HDL from one subject with no lipid with hypertriglyceridemia or combined In patients with diabetes, the presence and no glucose metabolism on secondary prevention of microvascular complications abnormalities. (17), suggesting that apoC1 might (retinopathy and nephropathy), was The cholesteryl ester transfer rate was become dysfunctional as a constitutive/ recorded. Three stages were used to calculated from the known specific endogenous CETP inhibitor in high-risk define nephropathy: 0 for no radioactivity of the HDL donors and the populations. nephropathy, 1 for microalbuminuria between30and300mgperday,and2 accumulation of radiolabeled Glycation is known to change side chains for proteinuria .300 mg per day. The cholesteryl esters in the LDL acceptors of reactive amino acids, essentially lysine estimated glomerular filtration rate was after the deduction of blank values and arginine, to form Schiff base and to calculated with the MDRD equation. from control mixtures that were reduce the isoelectric point of incubated at 378C without a plasma (pI) (19). Glycation is a consequence of This prospective single-center study was sample (12,22). hyperglycemia during diabetes. ApoC1 approved by our regional ethics committee. Written informed consent CETP mass concentration was measured contains nine lysine residues and three by a specific ELISA with TP1 anti-CETP arginine residues and is thus likely to be was obtained from all patients before inclusion in this study. antibodies as previously described (23). susceptible to glycation. CETP activity values were calculated as We hypothesized that glycation of apoC1 Plasma Preparation the ratio of the plasma cholesteryl ester might change its electrostatic properties, Blood samples were collected at transfer rate to the plasma CETP mass which are critical in its inhibitory effect, and inclusion and were centrifuged for concentration and were expressed as 1150 ApoC1 Glycation and CETP Inhibitory Property of ApoC1 Diabetes Care Volume 37, April 2014

nanomoles of 3H-CE transferred per from control subjects and patients with P values ,0.05 were considered milligram of CETP per hour. with T1D). statistically significant. Purification of ApoC1 by Chromatofocusing Measurement of Cholesteryl Ester RESULTS ApoC1 was purified from delipidated Transfer Activity by the Fluorescent CETP Activity Correlates With HDL apos by using the chromatofocusing Method ApoC1 Concentrations in Healthy method of Tournier et al. (24). For the in vitro analyses, CETP activity Normolipidemic-Normoglycemic fl was assessed by the uorescent method Control Subjects but Not in Patients Preparation of Lipoproteins as previously described (15). For the With T1D and T2D HDLs were isolated from plasma by measurement of cholesteryl ester Table 1 presents the characteristics of sequential flotation ultracentrifugation, transfer activity with apoC1, donor the subjects divided into three groups: according to their density, as previously liposomes (5 mL), acceptor VLDL (5 mL), healthy normolipidemic-normoglycemic described (12). and purified CETP were incubated in subjects, T1D patients, and T2D the presence of 250 pmol native apoC1 patients. Plasma TG were higher in T2D In Vitro Glycation of ApoC1, HDL, and or MG-treated apoC1. For the 6 CETP With Methylglyoxal patients than in control subjects (2.34 measurement of cholesteryl ester 6 , Methylglyoxal (MG) was used to glycate 1.79 vs. 0.86 0.24 mmol/L, P transfer activity with HDL, donor in vitro apoC1, HDL, and CETP. 0.001). TG levels in T1D patients were liposomes (5 mL) and purified CETP significantly higher than those in control MG reacts with the free amino groups of (5 mL) were incubated in the presence subjects (1.16 6 0.77 vs. 0.86 6 0.24 lysine and arginine (19). Thirteen (125 mL) of control HDL or HDL treated mmol/L, P , 0.0001), and significantly reactive groups are contained in 1 mol with MG (100 nmol cholesterol). lower than in T2D patients (1.16 6 0.77 apoC1, 112 reactive groups are vs. 2.34 6 1.79 mmol/L, P , 0.0001). contained in 1 mol HDL, and 41 reactive Agarose Gel Electrophoresis Plasma apoC1 concentrations were groups are contained in 1 mol CETP. The The electrophoretic mobility of native or significantly higher in T1D and T2D amount of MG used is represented as MG-treated HDL was determined by patients than in control subjects the ratio of the reactive groups in electrophoresis on 0.5% agarose gels (100.87 6 27.58, 96.7 6 28.28, and apoC1, HDL, and CETP that can interact (Paragon Lipo kit; Beckman 86.53 6 18.01 mg/L, respectively; P , with MG. For example, 50 mg apoC1 was Instruments) according to the method 0.005). Plasma apoC1 levels in T1D and treated with 0, 10, 25, 50, 100, and 500 of Sparks and Phillips (25). nmol MG corresponding to a ratio of 0:1, T2D patients were similar. 0.1:1, 0.25:1, 0.5:1, 1:1, and 5:1 of apoC1 Statistics CETP activity was significantly higher in reactive groups. Data are shown as means 6 SD or T1D and T2D patients than in control 6 6 The mixtures with apoC1 and HDL were percentages as indicated. Differences subjects (0.32 0.14, 0.43 0.13, and 6 3 incubated for 24 h at 378Canddialyzed between two groups were evaluated by 0.25 0.075 nmol H-CE/mg/h, , twice for 16 h against TBS. The mixtures the Student t test for continuous respectively; P 0.0001). In addition, with CETP were incubated for 1 h at variables. Differences between more CETP activity was higher in T2D patients , room temperature. than two groups for continuous than in T1D patients (P 0.0001) variables were analyzed by ANOVA. (Table 1). Isoelectric Analysis The Pearson correlation coefficients As shown in Fig. 1A and in Table 2, the Native apoC1, MG-treated apoC1, and were determined by linear regression plasma apoC1 concentration correlated HDL apoproteins from control subjects analysis. Statistical significance of the negatively with CETP activity in (n = 6) or patients with T1D (n =6)and correlation coefficients was normolipidemic-normoglycemic control protein pI standards (SERVA) were determined by the method of Fisher subjects (r = 20.309, P =0.005).In m diluted in 125 L hydration buffer and Yates. multivariate analysis, apoC1 (8 mol/L urea, 4% CHAPS, 20 mmol/L Multivariate analyses were done with concentration still correlated negatively dithiothreitol, 0.2% Bio-Lyte 3–10). fi b CETP activity as a dependent variable in and signi cantly with CETP activity ( = After overnight hydration of 7-cm-long 20.364, P = 0.001). In addition, in this ReadyStrip, pH 3–10 (Bio-Rad), at 50 V each group (control subjects, T1D, and T2D). The variables introduced into the population, the TG level correlated in a Protean IEF cell (Bio-Rad), fi model were age, BMI, HDL-C, LDL-C, TG, positively and signi cantly with CETP isoelectric focusing was conducted activity (b =0.255,P = 0.018). for 12 kV/h. Either the strips were and apoC1. In addition, HbA1c and stained with Coomassie Brilliant Blue treatment, which has been shown to In contrast to control subjects, no fi G-250 (for native or MG-treated potentially modify CETP activity, were signi cant relationship between CETP apoC1) or proteins were transferred introduced into the model in patients activity and apoC1 concentration was to a polyvinylidene fluoride membrane with diabetes. Multivariate analyses observed in T1D and T2D patients – for Western blotting with a rabbit anti- were done by stepwise multivariate (Fig. 1B C). human apoC1-immunoglobulin and linear regression. In the T1D group, univariate analysis an HRP-conjugated anti-rabbit The statistical analysis was done using revealed that CETP activity correlated antibody (Dako) (for HDL apoproteins SPSS software (Chicago, IL). Differences positively with HbA1c (P = 0.001), LDL-C care.diabetesjournals.org Bouillet and Associates 1151

fi Table 1—Characteristics of study population cholesteryl esters was again signi cantly , Control subjects T1D patients T2D patients higher in T1D patients with TG 1.7 mmol/L than in control subjects (0.29 6 n 83 70 113 0.12 and 0.25 6 0.07 nmol 3H-CE/mg/h, 6 6 6 b,c Age (years) 43.07 14.22 42.1 15.9 59.34 9.48 respectively; P = 0.022). In the subgroup Male sex, n (%) 47 (57) 39 (56) 61 (54) of T1D patients with plasma TG ,1.7 a b,c Weight (kg) 68.3 6 11.37 74.59 6 16.49 96.67 6 20.38 mmol/L, univariate analysis revealed BMI (kg/m2)23.586 3.67 25.66 6 5.34a 34.81 6 7.01b,c that CETP activity correlated positively Statin, n (%) 0 (0) 18 (25.7) 57 (50.4) with HbA1c (P = 0.03) and negatively Duration of diabetes (years) 20 6 12.5 12.7 6 9.5 with age (P = 0.037). In multivariate Retinopathy, n (%) 36 (51.4) 34 (30) analysis, CETP activity was Nephropathy, n (%) independently and positively associated Stage 0 53/68 (77.9) 51/109 (46.8) with HbA1c (b=0.274, P = 0.028) and Stage 1 10/68 (14.7) 41/109 (37.6) negatively with BMI (b =0.286,P = Stage 2 5/68 (7.4) 17/109 (15.6) 0.021) and statin therapy (b = 20.250, Treatment of diabetes, n (%) P = 0.044). In contrast, no relationship OAD 2 (2.9) 97 (85.8) Metformin 2 (2.9) 69 (61.1) between CETP activity and both apoC1 Sulfonylureas 0 (0) 69 (61.1) (b =0.08,P =0.516)andTG(b =0.153, a-Glucosidase inhibitors 0 (0) 2 (1.8) P = 0.245) was found in this subgroup. DPP4 inhibitors 0 (0) 12 (10.6) Finally, in order to assess the influence GLP-1 agonists 0 (0) 8 (7.1) of HbA1c on CETP activity, we divided therapy 70 (100) 65 (53.7) T1D patients into quartiles according to Long acting 0 (0) 37 (32.7) HbA1c. CETP activity was significantly Long acting plus short acting 61 (87.1) 28 (24.8) 6 Pump therapy 9 (12.9) 0 (0) higher in the fourth quartile (0.42 3 Insulin-OAD combination 2 (2.9) 51 (45.1) nmol H-CE/mg/h, HbA1c .9.6% [81 Glycemia (mmol/L) 5.24 6 0.44 9.69 6 4.41b 10.25 6 4.2b mmol/mol]) than in the first quartile (0.26 6 0.07 nmol 3H-CE/mg/h, HbA HbA1c 1c % ND 8.7 6 1.76 8.77 6 1.76 ,7.4% [57 mmol/mol], P , 0.0001). No mmol/mol 72 6 0.21 72 6 0.21 significant difference in TG levels was Creatinine (mmol/L) 86.42 6 13.63 78.3 6 37.37 90.07 6 29.28 observed between the first and the eGFR (mL/min) 81.23 6 13.4 101.09 6 30.83 77.49 6 24.92 fourth quartile. The ANOVA analysis for .60 79 62 83 CETP activity between quartiles was 30–60 4 6 30 strongly significant (F =6.913,P , 0.0001). 15–30 0 1 0 ApoC1 was significantly higher in the , 15 0 0 0 fourth quartile (107.6 mg/L) than in the 6 6 6 Total cholesterol (mmol/L) 4.72 0.89 4.94 1.26 4.74 1.18 first quartile (90.9 mg/L) (P = 0.045). LDL-C (mmol/L) 3.06 6 0.71 2.84 6 1.05 2.74 6 0.94a HDL-C (mmol/L) 1.48 6 0.41 1.62 6 0.46 1.08 6 0.25b,c In T2D patients, CETP activity correlated positively with total cholesterol (P = TG (mmol/L) 0.86 6 0.24 1.16 6 0.77a 2.34 6 1.79b,c 0.004), LDL-C (P = 0.04), and TG (P , ApoC1 mass (mg/L) 86.53 6 18.01 100.87 6 27.58b 96.7 6 28.28a 0.0001) and negatively with HDL-C (P , CETP mass (mg/L) 4.55 6 0.94 3.71 6 1b 2.82 6 0.76b,c 0.0001) (Table 2). Multiple linear regres- CETP activity (nmol 3H-CE/mg/h) 0.25 6 0.075 0.32 6 0.142b 0.43 6 0.135b,c sion analyses showed in T2D that CETP Data are means 6 SD, unless otherwise indicated. DPP4, dipeptidyl-peptidase 4; eGFR, activity was independently associated estimated glomerular filtration rate; GLP-1, -like peptide 1; OAD, oral antidiabetic drug. b a,bSignificantly different from control, P , 0.01, P , 0.0001, respectively. cSignificantly different with LDL-C ( = 0.204, P = 0.019), TG from T1D, P , 0.0001. (b =0.249,P = 0.015), and HDL-C (b = 20.345, P = 0.001). Thus, we hypothesize that glycation of apoC1 might change (P = 0.02), and TG (P , 0.0001) and is known to influence CETP activity its electrostatic properties and impair its negatively with HDL-C (P , 0.0001) (17,26), CETP activity in one subgroup of ability to inhibit CETP activity. and age (P = 0.019) (Table 2). In , T1D patients with plasma TG 1.7 Glycation of ApoC1 by MG Changes Its multivariate analysis, CETP activity was mmol/L was compared with CETP Electrostatic Charge and Impairs Its independently and positively associated activity in control subjects. Whereas b , Ability to Inhibit CETP Activity with the TG level ( = 0.411, P 0.0001) there was no significant difference Glycation of apoC1 in the presence of b and HbA1c ( =0.240,P = 0.022) and between control subjects and T1D MG led to the emergence of new acidic b negatively with statin therapy ( = patients with TG ,1.7 mmol/L in TG isoforms. A gradual shift of apparent pI 2 0.295, P =0.004). levels (0.86 6 0.24 mmol/L, n =83,vs. values from 8.3 for native apoC1 down As plasma TG were higher in T1D 0.89 6 0.27, n = 58, respectively; ns), to 4.0 was observed when apoC1 was patients than in control subjects and TG the ability of CETP to exchange treated with the highest amount of MG. 1152 ApoC1 Glycation and CETP Inhibitory Property of ApoC1 Diabetes Care Volume 37, April 2014

Figure 1—Relationship between apoC1 concentration and CETP activity in control subjects (A), T1D patients (B), and T2D patients (C). Correlation coefficients were calculated by Pearson test.

In accordance with previous studies, the was accompanied by a marked pretreatment of apoC1 with MG. addition of purified apoC1 to inhibition of the lipid transfer reaction MG-induced glycation of CETP produced a reconstituted mixtures containing (Fig. 2). The inhibitory potential of significant decrease in CETP activity in a isolated lipoprotein substrates and CETP human apoC1 was markedly affected by dose-dependent manner (P = 0.004). care.diabetesjournals.org Bouillet and Associates 1153

the proportion of apoC1 located Table 2—Correlations between CETP activity and age, BMI, glycemia, HbA1c, total cholesterol, HDL-C, LDL-C, TG, and ApoC1 in control subjects and T1D and T2D between 7.4 and 9.5 of the pI range patients was significantly lower than that in CETP activity (nmol 3H-CE/mg/h) control subjects (18.6 vs. 43.2%, rPrespectively; P = 0.0065), whereas the proportion between 3.5 and 4.2 Control subjects (n =83) of pI range was significantly higher Age 0.110 0.323 BMI 0.062 0.577 (18.1 vs. 5.9%, respectively; P = 0.0067). Total cholesterol 0.042 0.705 These results indicate that the LDL-C 0.135 0.225 electrostatic properties of apoC1 are HDL-C 20.163 0.141 modified in T1D. TG 0.176 0.112 ApoC1 20.309 0.005 T1D patients (n =70) CONCLUSIONS Age 20.279 0.019 In the current study, the negative BMI 0.199 0.098 correlation between apoC1 and CETP HbA1c 0.392 0.001 activity found in control subjects is Total cholesterol 0.192 0.114 shown here for the first time to be LDL-C 0.279 0.02 HDL-C 20.418 <0.0001 absent in T1D and T2D patients. As TG 0.460 <0.0001 glycation, a consequence of ApoC1 0.137 0.258 hyperglycemia in diabetes, is known to T1D patients with TG ,1.7 mmol/L (n =58) change isoelectrophoretic point of Age 20.275 0.037 proteins, we hypothesized and BMI 0.259 0.05 demonstrated that in vitro glycation of HbA1c 0.285 0.03 apoC1 modified its electrostatic Total cholesterol 0.174 0.192 properties and impaired its ability to LDL-C 0.245 0.064 HDL-C 20.181 0.174 inhibit CETP activity and that the TG 0.121 0.366 electrostatic properties of apoC1 are ApoC1 0.128 0.339 changed in patients with diabetes. T2D patients (n =113) As previously reported (16,17), we 2 Age 0.072 0.446 found a significant negative correlation BMI 20.020 0.831 between apoC1 concentration and HbA1c 0.143 0.134 Total cholesterol 0.271 0.004 CETP activity in normolipidemic- LDL-C 0.194 0.04 normoglycemic control subjects. These HDL-C 20.428 <0.0001 data confirm that apoC1 is a potent TG 0.386 <0.0001 endogenous inhibitor of CETP activity. ApoC1 0.049 0.608 One of our major results is that the Data are in boldface type when P , 0.05. inhibitory effect of apoC1 on CETP activity is abrogated in both T1D and T2D patients, as reflected by the loss of the negative correlation between Glycation of HDLs by MG Made Them Importantly, in vitro glycation of apoC1 specific CETP activity and apoC1 More Electronegative and Made Them by MG was shown here to abrogate its concentration in these populations. The Better Substrates for CETP CETP inhibitory effect and to change its major common abnormality in T1D and Because apoC1 is an inhibitor of CETP electrostatic charge properties. Because T2D is hyperglycemia. We hypothesized activity when associated with HDL, the glycation is a well-known consequence that hyperglycemia might be electrostatic properties of HDL and the of hyperglycemia in diabetes, we set out responsible for this loss of inhibitory ability of HDL to inhibit CETP activity to determine whether the electrostatic potential. The independent association were assessed after glycation with MG. charge of apoC1 in patients with between CETP activity and HbA1c MG-induced glycation of HDL diabetes was different from that in observed in T1D patients is in keeping produced a significant change in the healthy normolipidemic-normoglycemic with this hypothesis. On top of that, electrostatic properties of plasma HDL, subjects. CETP activity in the quartile of T1D which gradually became more patients with the highest HbA1c level electronegative as the MG Electrostatic Charge of Plasma ApoC1 was significantly greater than that in concentration increased (data not Is Abnormal in Patients With T1D the quartile of T1D patients with the shown). The ability of HDL treated with ApoC1 is known to have a pI value of lowest HbA1c level. The higher the MG to block the lipid transfer reaction 8.3. Accordingly, the pI of apoC1 in most degree of hyperglycemia, the greater was significantly weaker than that of the healthy control subjects ranged the loss of inhibitory potential of apoC1 achieved by native HDL (data not shown). between 7.4 and 9.5. In T1D patients, seemed to be. We did not find a 1154 ApoC1 Glycation and CETP Inhibitory Property of ApoC1 Diabetes Care Volume 37, April 2014

Figure 2—Effect of MG on isoelectrophoretic charge of apoC1 and on its ability to inhibit CETP. Effect of glycation of CETP with MG on CETP activity. Purified human CETP (5 mL) was incubated with apoC1 (1 mmol/L) treated with 0, 10, 25, 50, 100, and 500 nmol MG. CETP activity was expressed as compared with CETP activity measured without apoC1, which represents 100%. ***Significantly different from control, P , 0.0001.

significant correlation between HbA1c manner. Importantly, the decreasing diabetes might be different from those and CETP activity in T2D patients, but it electropositivity of apoC1 with of apoC1 from normolipidemic- seems likely that hypertriglyceridemia, increasing amounts of MG was normoglycemic subjects. Isoelectric which is highly present in this associated with a progressive loss of the analysis, conducted in vivo in the population, has a major effect on CETP CETP-inhibitory properties of apoC1. current study, showed that the activity and, thus, the effect of These findings are in accordance with electrostatic properties of apoC1 from hyperglycemia on CETP activity may earlier in vitro studies with acetylated T1D patients were different (reduced have been overwhelmed by that of apoC1, which linked the reduction in the electropositivity) from those in control hypertriglyceridemia. positive electrostatic charge of apoC1 subjects. Indeed, apoC1 from T1D fi ApoC1 is a highly basic protein with its impaired inhibitory activity on patients was signi cantly less basic than containing nine lysine residues and CETP (15). control apoC1. Pretreatment of apoC1 with MG was found to modify its pI three arginine residues. It has been The electrostatic properties of apoC1 value in a way similar to what is shown in shown that the inhibitory effect of were shown in vitro to be able to the current study to occur in T1D human apoC1 is a direct consequence of produce a significant change in HDL patients. its unique electrostatic properties, electronegativity (15), which is which impair HDL-CETP interactions recognized today as a leading factor that In the current study, CETP activity in (15). Glycation, a consequence of determines both the strength of CETP- both T1D and T2D patients was greater hyperglycemia in diabetes, is known to HDL interactions and the velocity of than that in control subjects as affect reactive amino acids of proteins, CETP-mediated lipid transfers (27–29). previously reported (30,31). CETP fi essentially lysine and arginine, and to The inhibition of CETP activity by apoC1 activity was signi cantly higher in T2D reduce the isoelectric point of proteins. is dependent, at least in part, on its than in T1D patients. This difference fi For the rst time, we investigated peculiar electrostatic properties, and may be partly explained by a whether glycation could change the significantly higher TG level in T2D, human apoC1 was found in earlier electrostatic properties of apoC1, and which directly stimulates CETP activity. studies to contribute significantly to the thus its ability to inhibit CETP activity, The strong and independent association overall surface charge potential of HDL and we studied the effect of in vitro between CETP activity and TG in T2D (15,16). In the current study, we showed glycation of human apoC1 with MG patients stresses the fact that that HDLs treated with MG were more (a protein glycating agent) on the hypertriglyceridemia is a major electronegative than native HDLs. The electrostatic and inhibitory properties determinant of CETP activity as glycation of positively charged amino of apoC1. MG interacts with lysine and previously reported (26). Although T1D acid residues of apoC1 may explain in arginine residues to create Schiff bases patients have plasma TG levels in the and adducts that decrease the pI of the part why glycated HDLs are more normal range, plasma TG levels were protein (19). As expected, the electronegative and therefore show a higher than those in control subjects, electrostatic properties of apoC1 were weaker ability to inhibit CETP activity. and plasma TG correlates significantly markedly changed by pretreatment with We hypothesized that the electrostatic with CETP activity. However, T1D MG and in a concentration-dependent properties of apoC1 from patients with patients with TG levels ,1.7 mmol care.diabetesjournals.org Bouillet and Associates 1155

showed significantly higher CETP mechanism underlying this increase government grant managed by the French activity than control subjects when their remains unclear, it shows that increased National Research Agency under the program “ ’ ” CETP activity in diabetic patients Investissements d Avenir with reference ANR- plasma TG levels were not different 11-LABX-0021/LipSTIC Labex. from those in control subjects. On top of occurs despite elevated apoC1 Duality of Interest. No potential conflicts of that, in this population, no correlation concentrations, stressing once again interest relevant to this article were reported. the fact that apoC1 is dysfunctional in was observed between CETP activity Author Contributions. B.B. carried out the and TG. Thus, the greater ability of CETP these patients. research, analyzed and interpreted data, to exchange cholesteryl esters between High CETP activity is frequently drafted the first manuscript, contributed to the lipoproteins in T1D patients is probably interpretation of the results, reviewed the observed in subjects with metabolic manuscript, and approved the final version of due to mechanisms other than disorders. These observations support themanuscript.T.G.andD.B.helpedwiththe increased TG levels, such as the view that a reduction in CETP study design, research, and interpretation of hyperglycemia. Indeed, in T1D patients, activity in this population might be data; contributed to the interpretation of the hyperglycemia correlated with CETP results; reviewed the manuscript; and antiatherogenic. However, despite a fi activity in both univariate and approved the nal version of the manuscript. clear favorable impact on the lipid J.-P.P.d.B. helped with the study design, multivariate analyses. profile, with a large increase in HDL-C contributed to the interpretation of the In this study, whereas CETP activity was levels and a specific reduction in dense results, reviewed the manuscript, and LDL particles (37), CETP inhibition approved the final version of the manuscript. shown to increase in patients with L.D. and J.-M.P. helped with data collection, diabetes, CETP activity was significantly therapy failed to demonstrate any fi fi contributed to the interpretation of the decreased by glycation of CETP in a signi cant ef cacy to reduce the results, reviewed the manuscript, and dose-dependent manner. Thus, the progression of atherosclerosis or the approved the final version of the manuscript. contribution of direct CETP glycation to occurrence of cardiovascular events L.L. and B.V. developed the study concept, supervised the study, contributed to the the observed changes is unlikely, and (38). This suggests that residual optimal CETP activity should be maintained in interpretation of the results, reviewed the this reinforced the potential role of manuscript, and approved the final version of glycation of apoC1 and its implication in order to preserve the antiatherogenic the manuscript. B.B. is the guarantor of this increasing CETP activity in diabetic properties associated with the work and, as such, had full access to all the physiological action of CETP (39). data in the study and takes responsibility for patients. the integrity of the data and the accuracy of As glycation is a consequence of In T1D patients, we would expect to find the data analysis. hyperglycemia, we hypothesize that low plasma HDL-C, as CETP activity is Prior Presentation. This study was presented lowering hyperglycemia may solve, at increased, but this was not the case. A in abstract form at the 81st Congress of the least in part, the loss of constitutive European Atherosclerosis Society, Lyon, France, slight increase in the level of HDL-C has CETP inhibition, thus maintaining 2–5 June 2013. been observed in T1D patients (32). optimal CETP activity in patients with Some studies have shown an increase in diabetes. References HDL2 (33,34) or in HDL3 (35). This 1. Malmendier CL, Lontie JF, Grutman GA, increase in plasma HDL-C could be the We showed that in vitro glycation of Delcroix C. Metabolism of apolipoprotein consequence of the elevated apoC1 impairs its ability to inhibit CETP C-I in normolipoproteinemic human (LPL)–to–hepatic activity. We suggest that the glycation of subjects. Atherosclerosis 1986;62:167–172 lipase (HL) ratio (increased LPL activity apoC1, which modifies its electrostatic 2. Westerterp M, de Haan W, Berbee´ JFP, and normal HL activity) (34). The properties and thus the electrostatic Havekes LM, Rensen PCN. Endogenous apoC-I increases hyperlipidemia in apoE- increased LPL activity observed in these properties of HDL particles, leads to increased binding of CETP to HDL and knockout mice by stimulating VLDL patients is probably due to peripheral production and inhibiting LPL. J Lipid Res hyperinsulinemia as a consequence of thus to increased CETP activity and 2006;47:1203–1211 cholesteryl ester transfer. We also the subcutaneous route of insulin 3. Havel RJ, Fielding CJ, Olivecrona T, Shore administration (34). showed in vivo that the electrostatic VG, Fielding PE, Egelrud T. activity properties of apoC1 are modified in of protein components of human very low The plasma concentration of apoC1 was patients with T1D and that apoC1 from density lipoproteins in the hydrolysis of fi found to be slightly though signi cantly diabetic patients has lost its ability to triglycerides by lipoproteins lipase from higherinT1DandT2Dpatientsthan different sources. Biochemistry 1973;12: inhibit CETP activity. Hyperglycemia 1828–1833 in control subjects. High apoC1 plays a major role in the loss of the 4. Conde-Knape K, Bensadoun A, Sobel JH, concentrations were reported in inhibitory potential of apoC1 on CETP dyslipidemic patients in one clinical Cohn JS, Shachter NS. Overexpression of activity. apoC-I in apoE-null mice: severe study (36) but not in another one (17). hypertriglyceridemia due to inhibition of The increase in apoC1 concentrations . J Lipid Res 2002;43:2136– in patients with diabetes may be Acknowledgments. The authors thank Philip 2145 explained by increased hepatic Bastable (University Hospital of Dijon) for 5. Berbee´ JFP, van der Hoogt CC, manuscript editing. production or decreased clearance of Sundararaman D, Havekes LM, Rensen PCN. apoC1. Kinetic studies of apoC1 are Funding. This work was supported by grants Severe hypertriglyceridemia in human from Universite´ de Bourgogne, Centre APOC1 transgenic mice is caused by apoC-I- needed to understand this rise in Hospitalier Universitaire de Dijon, Conseil induced inhibition of LPL. J Lipid Res 2005; apoC1 concentration. Although the Regional´ de Bourgogne, INSERM, and a French 46:297–306 1156 ApoC1 Glycation and CETP Inhibitory Property of ApoC1 Diabetes Care Volume 37, April 2014

6. Weisgraber KH, Mahley RW, Kowal RC, Herz 16. de Barros J-PP, Boualam A, Gautier T, et al. human cholesteryl ester transfer protein J, Goldstein JL, Brown MS. Apolipoprotein Apolipoprotein CI is a physiological activity. J Lipid Res 1996;37:1579–1590 C-I modulates the interaction of regulator of cholesteryl ester transfer 28. Sammett D, Tall AR. Mechanisms of with beta-migrating very protein activity in human plasma but not in enhancement of cholesteryl ester transfer – low density lipoproteins (beta-VLDL) and rabbit plasma. J Lipid Res 2009;50:1842 protein activity by lipolysis. J Biol Chem inhibits binding of beta-VLDL to low density 1851 1985;260:6687–6697 lipoprotein -related protein. J Biol 17. Pillois X, Gautier T, Bouillet B, et al. Chem 1990;265:22453–22459 29. Desrumaux C, Athias A, Masson D, Gambert Constitutive inhibition of plasma CETP by P, Lallemant C, Lagrost L. Influence of the 7. Sehayek E, Eisenberg S. Mechanisms of apolipoprotein C1 is blunted in electrostatic charge of lipoprotein particles inhibition by of dyslipidemic patients with coronary artery on the activity of the human plasma – apolipoprotein E-dependent cellular disease. J Lipid Res 2012;53:1200 1209 transfer protein. J Lipid Res metabolism of human -rich 18. Berbee´ JFP, Mooijaart SP, de Craen AJM, 1998;39:131–142 lipoproteins through the low density et al. Plasma apolipoprotein CI protects lipoprotein receptor pathway. J Biol Chem 30. Colhoun HM, Scheek LM, Rubens MB, – against mortality from infection in old age. et al. Lipid transfer protein activities in 1991;266:18259 18267 – J Gerontol A Biol Sci Med Sci 2008;63:122 type 1 diabetic patients without renal 8. Shachter NS, Ebara T, Ramakrishnan R, et al. 126 failure and nondiabetic control subjects Combined hyperlipidemia in transgenic 19. Schmitt A, Schmitt J, Munch¨ G, Gasic- and their association with coronary mice overexpressing human apolipoprotein Milencovic J. Characterization of advanced artery calcification. Diabetes 2001;50: Cl. J Clin Invest 1996;98:846–855 glycation end products for biochemical 652–659 fi 9. Jong MC, Dahlmans VE, van Gorp PJ, et al. In studies: side chain modi cations and 31. Riemens S, van Tol A, Sluiter W, Dullaart R. fl the absence of the low density lipoprotein uorescence characteristics. Anal Biochem Elevated plasma cholesteryl ester transfer receptor, human apolipoprotein C1 2005;338:201–215 in NIDDM: relationships with overexpression in transgenic mice inhibits 20. Friedewald WT, Levy RI, Fredrickson DS. -containing lipoproteins the hepatic uptake of very low density Estimation of the concentration of low- and phospholipid transfer protein. lipoproteins via a receptor-associated density lipoprotein cholesterol in plasma, Atherosclerosis 1998;140:71–79 protein-sensitive pathway. J Clin Invest without use of the preparative 1996;98:2259–2267 32. Dullaart RP. Plasma lipoprotein ultracentrifuge. Clin Chem 1972;18:499– abnormalities in type 1 (insulin-dependent) 10. Jong MC, Gijbels MJ, Dahlmans VE, et al. 502 diabetes mellitus. Neth J Med 1995;46:44– Hyperlipidemia and cutaneous 21. Dautin G, Soltani Z, Ducloux D, et al. 54 abnormalities in transgenic mice Hemodialysis reduces plasma overexpressing human apolipoprotein C1. 33. Eckel RH, Albers JJ, Cheung MC, Wahl PW, apolipoprotein C-I concentration making J Clin Invest 1998;101:145–152 Lindgren FT, Bierman EL. High density VLDL a better substrate for lipoprotein lipoprotein composition in insulin- 11. Jong MC, van Dijk KW, Dahlmans VE, et al. lipase. Kidney Int 2007;72:871–878 dependent diabetes mellitus. Diabetes Reversal of hyperlipidaemia in 22. Zeller M, Masson D, Farnier M, et al. High 1981;30:132–138 apolipoprotein C1 transgenic mice by serum cholesteryl ester transfer rates and adenovirus-mediated delivery of the 34. Kahri J, Groop PH, Viberti G, Elliott T, small high-density lipoproteins are low-density-lipoprotein receptor, but not Taskinen MR. Regulation of apolipoprotein associated with young age in patients with by the very-low-density-lipoprotein A-I-containing lipoproteins in IDDM. acute myocardial infarction. J Am Coll – receptor. Biochem J 1999;338:281–287 Diabetes 1993;42:1281 1288 Cardiol 2007;50:1948–1955 12. Gautier T, Masson D, de Barros JP, et al. 35. Winocour PH, Durrington PN, Ishola M, 23. Guyard-Dangremont V, Lagrost L, Gambert Human apolipoprotein C-I accounts for the Anderson DC. Lipoprotein abnormalities in P, Lallemant C. Competitive -linked ability of plasma high density lipoproteins insulin-dependent diabetes mellitus. immunosorbent assay of the human – to inhibit the cholesteryl ester transfer Lancet 1986;1:1176 1178 cholesteryl ester transfer protein (CETP). protein activity. J Biol Chem 2000;275: 36. Cohn JS, Tremblay M, Batal R, et al. Plasma Clin Chim Acta 1994;231:147–160 37504–37509 kinetics of VLDL and HDL apoC-I in 24. Tournier JF, Bayard F, Tauber JP. Rapid 13. Gautier T, Masson D, Jong MC, et al. normolipidemic and hypertriglyceridemic purification and activity of apolipoprotein – Apolipoprotein CI deficiency markedly subjects. J Lipid Res 2002;43:1680 1687 C1 on the proliferation of bovine vascular augments plasma lipoprotein changes 37. Guerin M, Le Goff W, Duchene E, et al. endothelial cells in vitro. Biochim Biophys mediated by human cholesteryl ester Inhibition of CETP by torcetrapib Acta 1984;804:216–220 transfer protein (CETP) in CETP transgenic/ attenuates the atherogenicity of ApoCI-knocked out mice. J Biol Chem 2002; 25. Sparks DL, Phillips MC. Quantitative postprandial TG-rich lipoproteins in type IIB 277:31354–31363 measurement of lipoprotein surface charge hyperlipidemia. Arterioscler Thromb Vasc by agarose gel electrophoresis. J Lipid Res – 14. Gautier T, Masson D, Jong MC, et al. Biol 2008;28:148 154 1992;33:123–130 Apolipoprotein CI overexpression is not a 38. Hooper AJ, Burnett JR. Dalcetrapib, a relevant strategy to block cholesteryl ester 26. de Vries R, Perton FG, Dallinga-Thie GM, cholesteryl ester transfer protein transfer protein (CETP) activity in CETP et al. Plasma cholesteryl ester transfer is a modulator. Expert Opin Investig Drugs transgenic mice. Biochem J 2005;385:189– determinant of intima-media thickness in 2012;21:1427–1432 195 type 2 diabetic and nondiabetic subjects: 39. Villard EF, El Khoury P, Duchene E, et al. role of CETP and triglycerides. Diabetes 15. Dumont L, Gautier T, de Barros J-PP, et al. Elevated CETP activity improves 2005;54:3554–3559 Molecular mechanism of the blockade of plasma cholesterol efflux capacity from plasma cholesteryl ester transfer protein by 27. Masson D, Athias A, Lagrost L. Evidence for human macrophages in women. its physiological inhibitor apolipoprotein CI. electronegativity of plasma high density Arterioscler Thromb Vasc Biol 2012;32: J Biol Chem 2005;280:38108–38116 lipoprotein-3 as one major determinant of 2341–2349