Lipid transfer to HDL in type-2 diabetic patients: associations with microalbuminuria, statin, and insulin

Abstracts

FUNDAMENTO: O diabete melito tipo 2 (DM2) é um fator de risco isolado para coronariopatia, principalmente quando associado à microalbuminúria (MA). Alterações estruturais e funcionais das lipoproteínas não são totalmente esclarecidas nesse contexto. OBJETIVO: Avaliar a transferência de lípides para HDL (T) em pacientes DM2 e a associação com a presença da MA e com o tratamento com estatina ou insulina. MÉTODOS: Estudamos 33 pacientes com DM2 e 34 controles pareados para idade. Uma nanoemulsão lipídica artificial radiomarcada com ³H-Triglicéride (TG) e 14C-colesterol livre (CL) ou ³H-colesterol éster (CE) e 14C-fosfolípide (FL) foi incubada com plasma. A nanoemulsão e as lipoproteínas foram precipitadas, exceto a HDL, que teve sua radioatividade contada. RESULTADOS: A TFL (%) foi maior no grupo com DM2 que no grupo-controle (25,2±3,2 e 19,7±3,2 respectivamente; p < 0,001), assim como a TCL (%): 9,1±2,7 e 6,3±1,5 respectivamente; p < 0,001. O diagnóstico de MA não se associou a mudanças da propriedade de transferência. O uso da insulina associou-se à menor TFL (%): 23,5±2,1 contra 26,1±3,3; p = 0,018. Já o uso da estatina associou-se à queda de todas - TCE (%): 3,5±0,9; TFL (%):23,8±2,0; TTG (%): 3,9±0,8; TCL (%):7,4±1,3 - quando comparado ao grupo que não usava estatina (TCE (%):5,9±2,4; TFL (%):26,9±3,6; TTG (%):6,4±2,2; TCL (%):11,1±2,6). CONCLUSÃO: O DM2 aumentou a transferência de lípides de superfície para HDL, enquanto o uso de estatina diminuiu todas as transferências de lípides. A presença de MA não se associou às alterações das transferências de lípides.

Colesterol HDL; lipoproteínas; diabete melito tipo 2; insulina; inibidores de hidroximetilglutaril-CoA redutase; albuminúria; nefropatias diabéticas


BACKGROUND: Type-2 diabetes mellitus (T2DM) is an isolated risk factor for coronary artery disease, especially when associated with microalbuminuria (MA). Structural and functional changes in lipoproteins have not yet been fully elucidated in this context. OBJECTIVE: To assess lipid transfer (T) to HDL in type-2 diabetic patients and its association with microalbuminuria and treatment with statins or insulin. METHODS: Thirty-three patients with type-2 diabetes mellitus and 34 age-matched control subjects were studied. A synthetic cholesterol-rich nanoemulsion radiolabeled with ³H- triglycerides (TG) and 14C-free cholesterol (FC) or ³H- cholesteryl ester (CE) and 14C-phospholipids (PL) was incubated with plasma. Both the nanoemulsion and lipoproteins were precipitated, except for HDL, which was counted for radioactivity. RESULTS: PLT (%) was higher in the T2DM group than in the control group (25.2 ± 3.2 and 19.7 ± 3.2 respectively; p < 0.001), as was free cholesterol (% FC): 9.1 ± 2.7 and 6.3 ± 1.5 respectively; p < 0.001. The diagnosis of microalbuminuria (MA) was not associated with changes in lipid transfers. Insulin therapy was associated with lower PLT rates: 23.5 ± 2.1 versus 26.1 ± 3.3; p = 0.018. Statin therapy, in turn, was associated with a drop in all lipid transfers - CET 3.5 ± 0.9; PLT: 23.8 ± 2.0; TGT: 3.9 ± 0.8; FCT: 7.4 ± 1.3 - as compared to the group that was not on statin therapy (CET: 5.9 ± 2.4; PLT: 26.9 ± 3.6; TGT: 6.4 ± 2.2; FCT: 11.1 ± 2.6). CONCLUSION:Type-2 diabetes mellitus increased lipid transfer to HDL particles, whereas statin therapy decreased all lipid transfers. The presence of MA was not associated with changes in lipid transfer.

Cholesterol, HDL; lipoproteins; diabetes mellitus, type 2; insulin; hydroxymethylglutaryl-CoA reductase inhibitors; albuminuria; diabetic nephropathies


FUNDAMENTO: La diabetes mellitus tipo 2 (DM2) es un factor de riesgo aislado para coronariopatía, principalmente cuando asociado a la microalbuminuria (MA). Alteraciones estructurales y funcionales de las lipoproteínas no están totalmente aclaradas en ese contexto. OBJETIVO: Evaluar no sólo la transferencia de lípidos hacia HDL (T) en pacientes DM2, sino también la asociación tanto con la presencia de la MA como con el tratamiento con estatina o insulina. MÉTODOS: Estudiamos a 33 pacientes con DM2 y 34 controles pareados para edad. Se incubó con plasma una nanoemulsión lipídica artificial radiomarcada con ³H-Triglicérido (TG) y 14C-colesterol libre (CL) o ³H-colesterol esterificado (CE) y 14C-fosfolípido (FL). Se procedió a la precipitación de la nanoemulsión y de las lipoproteínas, con excepción de la HDL, que tuvo su radioactividad contada. RESULTADOS: El valor de TFL (%) resultó mayor en el grupo con DM2 en confrontación con el grupo-control (25,2±3,2 y 19,7±3,2 respectivamente; p < 0,001); la TCL (%), por su vez, obtuvo los siguientes resultados: 9,1±2,7 y 6,3±1,5 respectivamente; p < 0,001. El diagnóstico de MA no se asoció a cambios de la propiedad de transferencia. El uso de la insulina se asoció al menor valor de TFL (%): 23,5±2,1 vs 26,1±3,3; p = 0,018. Ya el uso de la estatina se asoció a la baja del valor de todas las lipoproteínas - TCE (%): 3,5±0,9; TFL (%):23,8±2,0; TTG (%): 3,9±0,8; TCL (%):7,4±1,3 - si comparado al grupo que no usaba estatina (TCE (%):5,9±2,4; TFL (%):26,9±3,6; TTG (%):6,4±2,2; TCL (%):11,1±2,6). CONCLUSIONES: El DM2 aumentó la transferencia de lípidos de superficie hacia HDL, mientras que el uso de estatina disminuyó todas las transferencias de lípidos. La presencia de MA no se asoció a las alteraciones de las transferencias de lípidos.

Colesterol HDL; lipoproteínas; diabetes mellitus tipo 2; insulina; inhibidores de hidroximetilglutaril-CoA reductase; albuminuria; nefropatías diabéticas


ORIGINAL ARTICLE

Lipid transfer to HDL in type-2 diabetic patients: associations with microalbuminuria, statin, and insulin

Gilson Soares Feitosa-Filho; Talita de Mattos Seydell; Alina Coutinho Rodrigues Feitosa; Raul Cavalcante Maranhão; José Antônio Franchini Ramires

Instituto do Coração (InCor), Hospital do Coração da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP), São Paulo, SP - Brazil

Mailing address

SUMMARY

BACKGROUND: Type-2 diabetes mellitus (T2DM) is an isolated risk factor for coronary artery disease, especially when associated with microalbuminuria (MA). Structural and functional changes in lipoproteins have not yet been fully elucidated in this context.

OBJECTIVE: To assess lipid transfer (T) to HDL in type-2 diabetic patients and its association with microalbuminuria and treatment with statins or insulin.

METHODS: Thirty-three patients with type-2 diabetes mellitus and 34 age-matched control subjects were studied. A synthetic cholesterol-rich nanoemulsion radiolabeled with 3H- triglycerides (TG) and 14C-free cholesterol (FC) or 3H- cholesteryl ester (CE) and 14C-phospholipids (PL) was incubated with plasma. Both the nanoemulsion and lipoproteins were precipitated, except for HDL, which was counted for radioactivity.

RESULTS: PLT (%) was higher in the T2DM group than in the control group (25.2 ± 3.2 and 19.7 ± 3.2 respectively; p < 0.001), as was free cholesterol (% FC): 9.1 ± 2.7 and 6.3 ± 1.5 respectively; p < 0.001. The diagnosis of microalbuminuria (MA) was not associated with changes in lipid transfers. Insulin therapy was associated with lower PLT rates: 23.5 ± 2.1 versus 26.1 ± 3.3; p = 0.018. Statin therapy, in turn, was associated with a drop in all lipid transfers - CET 3.5 ± 0.9; PLT: 23.8 ± 2.0; TGT: 3.9 ± 0.8; FCT: 7.4 ± 1.3 - as compared to the group that was not on statin therapy (CET: 5.9 ± 2.4; PLT: 26.9 ± 3.6; TGT: 6.4 ± 2.2; FCT: 11.1 ± 2.6).

CONCLUSION:Type-2 diabetes mellitus increased lipid transfer to HDL particles, whereas statin therapy decreased all lipid transfers. The presence of MA was not associated with changes in lipid transfer.

Key words: Cholesterol, HDL; lipoproteins; diabetes mellitus, type 2; insulin; hydroxymethylglutaryl-CoA reductase inhibitors; albuminuria; diabetic nephropathies.

Introduction

Type-2 diabetes mellitus (T2DM) is a major cardiovascular risk factor, and atherosclerosis is the most common cause of death in diabetic patients. The dyslipidemia of T2DM is characterized by hypertriglyceridemia, decreased HDL-cholesterol, and increased small dense LDL particles1,2. Statins significantly decrease the risk of cardiovascular events in T2DM pacients3-5. The presence of microalbuminuria increases the risk of coronary artery disease6,7.

Lipoprotein are composed of different amounts of triglycerides and esterified cholesterol in their hydrophobic core and phospholipids and free cholesterol on their amphipatic surface, where apolipoproteins are located. HDL has antiatherosclerotic activity, the mechanism of which is not entirely understood. The most likely hypothesis is reverse cholesterol transport. Nevertheless, other mechanisms, such as anti-inflammatory, antithrombotic, and antioxidant properties8,9, seem to play a role in the protective effect of HDL.

Reverse cholesterol transport (RCT) is the process by which excess cholesterol is removed from peripheral tissues to the liver for subsequent excretion. Lecithin:cholesterol acyltransferase (LCAT) contributes to this transport by catalyzing the esterification of cholesterol, thus allowing it to be transferred to the hydrophobic core of the HDL particle.

Plasma lipoproteins exchange lipids constantly, a process facilitated by transfer proteins, such as cholesteryl ester transfer protein (CETP), which mediates the exchange of cholesterol esters, triglycerides, and phospholipids between lipoproteins10, and phospholipid transfer protein (PLTP), which mediates phospholipid and cholesterol transfer from other triglyceride-rich lipoproteins to HDL11,12.

Plasma PLTP13,14 and CETP15,16 activities seem to be higher in type-2 diabetic patients. The role of microalbuminuria in transfer protein activities has yet to be defined17. Some studies, however, show that the use of statin18-21 and insulin22-26 decreases plasma CETP and PLTP.

Lipid transfers depend on several other factors as well. Plasma concentration of each lipoprotein subclass influences these exchanges, since they are brought about by collisions between lipoprotein particles. The lipid and protein content of the particle and concentrations of several other plasma proteins may play a role in this process.

The complex relationship between lipid transfer and atherogenesis remains unclear. Lipid transfer plays a critical role in plasma HDL metabolism, by constantly remodeling lipoprotein particles and, thereby, interfering with their many functional aspects.

The purpose of this study was twofold: first, to assess, in T2DM patients, the simultaneous transfer to HDL of the four major lipids that make up its structure, namely, free cholesterol, cholesterol ester, triglycerides, and phospholipids; and second, to ascertain the influence of microalbuminuria, as well as that of statin or insulin therapy, on lipid transfer.

Methods

Thirty-three volunteer patients with type-2 diabetes mellitus were recruited from the outpatient services of the Hospital das Clínicas Heart Institute of the University of São Paulo Medical School (InCor - HCFMUSP) and the Diabetes Outpatient Clinic of the Division of Diabetes of the Discipline of Endocrinology and Metabolism of the Hospital das Clínicas of the University of São Paulo Medical School. Patients taking protease inhibitors, immunosuppressive agents, or corticosteroids were excluded from the study, as were transplant recipients and patients with liver cirrhosis, neoplasias, thyroid disease, or serum creatinine > 1.5 mg/dL. All patients signed an informed consent form. Blood and urine samples were collected at the Laboratory of Lipid Metabolism of InCor. HDL functional assays (lipid transfer) were performed at the same laboratory. Urine samples were tested at Hospital das Clínicas Central Laboratory, and blood samples at the InCor laboratory.

Thirty-four age-matched subjects were selected from the database of the Laboratory of Lipid Metabolism to serve as control group. No subject in this group had any comorbidity or was taking any medication. Those with fasting plasma glucose equal to or greater than 100 mg/dl were excluded.

All participants were interviewed about their medical history, with particular emphasis on past conditions and current medications. Laboratory tests were performed on a blood sample and two or three urine samples collected on different days to measure the urinary albumin/creatinine ratio.

Serial laboratory tests

The following parameters were measured in diabetic and control subjects: total cholesterol (TC), lipoprotein fractions (VLDL, LDL, and HDL), and fasting plasma glucose - in serum samples obtained after 12 hours of fasting - using the enzymatic colorimetric method (COD-PAD; Labtest). LDL-cholesterol was quantified using the Friedewald formula27: LDL-C = TC-(VLDL-C+HDL-C), in mg/dL, where VLDL-C is estimated by dividing triglyceride value by 5. Glycated hemoglobin (HbA1c) was measured in whole blood by immunoturbidimetric assay at the laboratory of InCor-HCFMUSP. The presence or absence of microalbuminuria was determined in first-morning spot urine samples by the nephelometric method at the Hospital das Clínicas Central Laboratory. Urine creatinine was measured in the same sample, and the albumin-creatinine ratio was expressed in mcg/mg of creatinine. Patients with urinary albumin-creatinine ratio less than 30 mcg/mg were considered normoalbuminuric, whereas those with urinary albumin-creatinine ratio between 30 and 300 mcg/mg were considered microalbuminuric, according to the American Diabetes Association criteria28.

Urine samples were collected on different days, outside of menses for women, from patients with no symptoms of urinary tract infection. If the results of the two tests did not agree, a third sample was requested.

LDE as an investigative tool

In earlier studies, the Laboratory of Lipid Metabolism of the Heart Institute (InCor-HCFMUSP) reproduced LDL metabolism using a synthetic emulsion with a lipid composition resembling that of the native LDL (LDE)29. The primary purpose of these studies was to use LDE to gain further insight into dyslipidemias. LDE is protein-free, but when it comes into contact with native lipoproteins it acquires apo E, which may be recognized by LDL receptors and, thereby, taken up by the cell29. This emulsion was prepared according to the technique described by Ginsburg et al30 and modified by Maranhão et al29.

Transfer of free cholesterol, cholesterol ester, triglycerides, and phospholipids from lde to hdl

Blood samples were collected after a 12-hour fast in EDTA-containing tubes (1.5 g/l), and plasma was obtained after centrifugation at 3.000 rpm for 10 minutes in a Sorval RT7 centrifuge. Two 200-µ plasma samples were incubated with 50 µl of LDE each for 60 minutes at 37°C in a Gyromax 706R orbital shaker and agitated at 40 rpm. Each 50 µl of LDE was radiolabeled with 3H- cholesteryl ester (3H-CE) and 14C-phospholipids (14C-PL) or 3H- triglycerides (3H-TG) and 14C-free cholesterol (14C-FC). After incubation, 250 µl of precipitating reagent for apo-B-containing lipoproteins (dextran sulfate 0.2%/ MgCl2 3M, v/v) was added to the tubes. The resulting mixtures were vortexed for 30 seconds and centrifuged for 10 minutes at 3.000 rpm. The HDL fraction was obtained after precipitation of the nanoemulsion, together with apo B-containing lipoproteins, with 250 µl of dextran/MgCl2 (0.2% Dextran and 0.3 mol/L MgCl2). Aliquots (250-µl ) of supernatant containing HDL were transferred with a pipette to scintillation vials containing 5.0 of scintillation fluid (Ultima Gold, PerkinElmer, Boston, USA), and radioactivity was measured on a beta counter (Liquid Scintillation Analyzer, Packard 1600 TR, Palo Alto, CA) using the Plus 5.01 software (Diamond Computers) to detect 14C and 3H in the samples. Sample blank was a mixture of 200 µl TRIS-HCl buffer and 50 µl of LDE, which was incubated and precipitaded as described previously. Total radioactivity in each sample was measured in 200 µl of plasma with 50 µl of LDE, followed by incubation, but without precipitating reagent. Lipid transfer from LDE to plasma HDL was expressed as percentage (%) of total radioactivity.

Statistical analysis

Statistical analysis was performed using SPSS 13.0 for Windows (Statistical Package for Social Sciences). Continuous variables were expressed as means and standard deviations, and categorical variables as percentages and absolute values.

The Kolmogorov-Smirnov test was performed for normal distribution. As variables were non-normally distributed, nonparametric tests were used. Differences between categorical variables were assessed by Fisher's exact test or the chi-square test.

Diabetic patients were compared with their controls, in addition to subgroups of diabetic patients, regarding the presence of microalbuminuria and use of statin and insulin. The Mann-Whitney test was performed to compare independent continuous variables, and Spearman's nonparametric test was used for correlations. Two tailed p values < 0.05 were considered statistically significant.

Results

Table 1 summarizes clinical data, plasma lipid composition, and glucose levels in T2DM patients and control subjects. Mean age of the T2DM group (55 ± 9) did not differ significantly from that of the control group.

No difference was found in plasma total cholesterol, LDL-C, and HDL-C between the T2DM group and the control group, p > 0.05. Triglycerides levels were 38% higher (p < 0.01) in the T2DM group than in the control group. Fasting plasma glucose was 44% higher in the T2DM group (p < 0.001). Body mass index was 19% higher among T2DM patients (p < 0.001).

Table 2 shows lipid values (cholesterol ester, phospholipids, triglycerides, and free cholesterol) transferred from the nanoemulsion to HDL in diabetic patients and control subjects.

There was no difference in cholesterol ester and triglyceride transfer from the nanoemulsion to HDL between diabetic patients and control subjects. As for free cholesterol and phospholipid transfers, these were approximately 30% and 22% higher in the diabetic group than in the control group, respectively (p < 0.001).

Among diabetic patients, no significant differences were found regarding the presence or not of microalbuminuria. However, mean age was 13% higher in the microalbuminuric than in the non-microalbuminuric group (p = 0.008). Age, gender, hypertension prevalence, body mass index, use of statins or insulin, plus lipid and glucose levels did not differ between both groups.

Comparing the subgroups of microalbuminuric and normoalbuminuric diabetic patients aged over 55, it was possible to compare groups within the same age range. Once more, there was no significant difference in lipid transfer to HDL between these groups (Table 3).

Using the urinary albumin-to-creatinine ratio as a continuous variable, a relationship was established between all variables by the Spearman correlation test. A higher urinary albumin/creatinine ratio was positively correlated with age and glycated hemoglobin and negatively correlated with free cholesterol transfer (Table 4).

Regarding insulin therapy, age, gender, body mass index, hypertension prevalence, plus lipid and glucose levels were similar in the insulin and non-insulin treated groups. The frequency of statin use was also similar in both groups. Insulin therapy was associated with a 10% decrease in phospholipid transfer to HDL (p = 0.018) (Figure 1).


With respect to statin therapy, transfer of all lipids to HDL was significantly lower in the group receiving this drug class than in the group that was not (Figure 2). Among T2DM patients, transfer of cholesterol ester, phospholipids, triglycerides, and free cholesterol was, respectively, 41%, 12%, 39%, and 33% lower in those who were on statins than in those who were not.


Discussion

The contribution of HDL-associated proteins to the generation, maturation, and reverse transport of cholesterol has been extensively studied. Cholesteryl ester transfer protein (CETP) modulates HDL levels and composition by mediating the exchange of neutral lipids (esterified cholesterol and triglycerides) between HDL and triglyceride-rich lipoproteins, in addition to promote phospholipid transfer to HDL. This protein also releases apolipoproteins from HDL in the presence of fatty acids31,32. Plasma phospholipid transfer protein (PLTP) itself can release apolipoproteins from HDL33. Cholesterol ester in HDL particles may originate from two sources: 1) esterification of HDL-free cholesterol, and 2) cholesterol ester of other lipoprotein34,35.

PLTP acts by transferring surface phospholipids to HDL and displacing apo AI from the particle's surface36,37. An important PLTP-mediated product of HDL particles are the small, lipid-poor pre-β-HDLs that act as initial acceptors of free cholesterol38,39. Apo E (but not apo AI) is capable of converting inactive PLTP into its active form40. This is a major characteristic that must be kept in mind, since LDE is devoid of apolipoproteins and acquires apo E when comes into contact with the blood.

This study provides a simple and fast manner to estimate HDL acceptor activity of all lipids using a single methodology. It allowed us to demonstrate that the uptake of phospholipids and free cholesterol by HDL is greater in diabetic patients, as compared to control subjects.

The finding of increased phospholipid transfer in diabetic patients, suggesting higher PLTP activity, is consistent with some reports in the literature13,14. Earlier studies have also shown an increase in plasma cholesteryl ester transfer15,16

The relationship between microalbuminuria and dyslipidemia in diabetic patients has been well explored. In this study, however, no differences were found in lipid transfer to HDL between normoalbuminuric and microalbuminuric T2DM patients. In the literature, no change in CETP activity was reported in microalbuminuric patients17.

Phospholipid transfer to HDL was lower in insulin-treated than in non-insulin-treated patients, with no changes in the other three lipids. PLTP is responsible for most phospholipid transfer to HDL and, to a lesser extent, facilitates the influx of free cholesterol to this lipoprotein. The present study suggests a possible correlation between insulin and PLTP activity. Most studies in the literature, though not all, that sought to investigate the relationship between insulin and PLTP activity have shown inhibition of the latter22-26.

Statin therapy was found to reduce the uptake of all lipids by HDL. Other studies have demonstrated that it decreases plasma CETP activity by three different mechanisms: 1) reducing plasma CETP mass18, 2) reducing lipoproteins with which HDL interacts19, and 3) possibly reducing CETP gene expression20. In a substudy of the DALI trial, statin therapy reduced plasma PLTP activity, despite increasing PLTP mass21. In the present study, the lipid acceptor activity of HDL was assessed. The other direction of lipid exchange between lipoproteins was not measured: the amount of lipids from HDL that is donated to the other lipoproteins is not known. Paradoxically, HDL acceptor activity was directly associated with risk factors of atherosclerosis, and statin therapy decreased all lipid transfer to HDL. This may be explained by the fact that the HDL with transfer characteristics that are more compatible with good markers is associated with less acquisition and loss of lipids and, therefore, would be a more stable HDL.

This study provides additional data regarding the complex and still incompletely understood mechanism of lipid exchange between lipoproteins. It also demonstrates that the use of insulin and statins is associated with functional aspects of HDL, as well as with its transfer proteins.

Study limitations

Diabetes mellitus is a condition that may have a broad clinical spectrum. An attempt was made to standardize the study population, excluding diabetic patients who were receiving exclusively non-pharmacological therapy, as well as those with evidence of renal failure or macroalbuminúria.

In spite of this, there was a degree of heterogeneity in the diabetic sample, in that diabetes mellitus was more advanced or more associated with comorbidities in some patients than in others. Moreover, part of the study sample was using insulin, statins, or anti-hypertensive drugs.

Ideally, any medication that might interfere with lipid metabolism or microalbuminuria should have been discontinued. That was not the case. Lipid measurements following discontinuation of statin therapy, therefore, were not performed, because we believe that such discontinuation would lead to a transient change in lipid levels in a population with cardiovascular risk factors without conferring any benefit. The same is true regarding anti-hypertensive and hypoglycemic drugs.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Sources of Funding

This study was funded by CNPq (the National Council of Technological and Scientific Development), and FAPESP (the São Paulo State Research Foundation).

Study Association

This article is part of the doctoral dissertation submitted by Gilson Soares Feitosa-Filho, from Faculdade de Ciências Médicas da Universidade do Estado do Rio de Janeiro.

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Publication Dates

  • Publication in this collection
    06 Apr 2009
  • Date of issue
    Feb 2009

History

  • Reviewed
    25 Feb 2008
  • Received
    16 Jan 2008
  • Accepted
    04 Mar 2008
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