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Preβ1-HDL is elevated in the fasting state, but markedly reduced postprandially in poorly controlled type 2 diabetic patients
Clinica Chimica Acta 401 (2009) 57–62
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n c h i m
Preβ1-HDL is elevated in the fasting state, but markedly reduced postprandially in poorly controlled type 2 diabetic patients Satoshi Hirayama a, Takako Ito b, Osamu Miyazaki c, Takashi Kamimura b, Osamu Hanyu b, Utako Seino d, Seiki Ito b, Yoshifusa Aizawa b, Takashi Miida a,⁎ a
Department of Clinical Laboratory Medicine, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan Division of Endocrinology and Metabolism, Niigata University, Niigata, Japan Sekisui Medical, Tsukuba Research Institute, Ibaraki, Japan d Division of Clinical Laboratory Medicine, Niigata University, Niigata, Japan b c
a r t i c l e
i n f o
Article history: Received 7 March 2008 Received in revised form 26 October 2008 Accepted 7 November 2008 Available online 17 November 2008 Keywords: Prebeta-HDL Postprandial change CETP LCAT
a b s t r a c t Background: Preβ1-HDL is a minor HDL subfraction that is an initial acceptor of cellular free cholesterol. Preβ1-HDL is elevated in hypertriglyceridemia, which is exaggerated with postprandial hyperglycemia. We investigated whether the preβ1-HDL concentration changes postprandially in type 2 diabetic patients and blood glucose (BG) control reduces this change. Methods: We examined 9 healthy controls and 20 diabetic patients with poor BG control. Seven blood samples (30 min before and 2 h after each meal, and at midnight) were obtained daily in the poor (poor-GC: n = 20) and improved (imp-GC: n = 11) glycemic control phases of diabetic patients after intensive insulin therapy and a low-calorie diet. Results: The preβ1-HDL concentration did not change postprandially in the controls. However, the fasting preβ1-HDL concentration in the poor-GC phase was 28.3% higher than in the controls (25.4 ± 6.8 vs 19.8 ± 6.9 mg/l ApoAI, p b 0.05) and decreased markedly after breakfast (20.9 ± 7.7 mg/l ApoAI, p b 0.01). In the impGC phase, the preβ1-HDL concentration showed no morning surge, as in the controls. Conclusions: Type 2 diabetic patients in the poor-GC phase have high preβ1-HDL levels in the morning, followed by a gradual reduction until midnight. BG control diminishes this postprandial change. Glucose metabolism may be involved in modulating reverse cholesterol transport in type 2 diabetic patients. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The incidence of type 2 diabetes has increased in Japan, as the lifestyle has been somewhat ‘Westernized’ over the last 60 y [1]. Although strict glycemic control reduces diabetic microangiopathy [2,3], it does not significantly reduce cardiovascular disease (CVD) [4], which remains the leading cause of death in diabetic patients [5,6]. Diabetic patients often have high triglyceride (TG) and low HDL-cholesterol (HDL-C) levels [7]. Patients with such lipoprotein profiles are at higher risk of CVD than normolipidemic diabetics [5,8]. Furthermore, hypertriglyceridemia is often exaggerated in the postprandial state, which is another independent risk factor for CVD [9].
Abbreviations: CVD, Cardiovascular disease; TG, Triglyceride; HDL-C, HDL-cholesterol; ApoAI, Apolipoprotein AI; TGRL, Triglyceride rich lipoprotein; LPL, Lipoprotein lipase; CETP, Cholesteryl ester transfer protein; LCAT, Lecithin cholesterol acyltransferase. ⁎ Corresponding author. Tel.: +81 3 5802 1104; fax: +81 3 5684 1609. E-mail address: [email protected] (T. Miida). 0009-8981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.11.010
HDL removes excess cholesterol from peripheral tissues and transports it to the liver for excretion via the bile (reverse cholesterol transport) [10]. Since HDL particles vary in size, composition, and function, they are classified into several subfractions by different procedures [11–17]. Preβ1-HDL is a minor apolipoprotein AI (ApoAI)containing subfraction, which was first identified by native twodimensional gel (2D-gel) electrophoresis. Preβ1-HDL is primarily composed of ApoAI and phospholipids (surface components of lipoproteins) and it has little core lipid. Experimental data indicate that preβ1-HDL serves as an initial acceptor of cellular cholesterol [15]. The fasting preβ1-HDL concentration is elevated in hypertriglyceridemia [12]. This may reflect enhanced production of preβ1-HDL from αmigrating, TG-rich HDL particles by hepatic lipase [18,19], or the surface remnants of TG-rich lipoproteins (TGRL) hydrolyzed by lipoprotein lipase (LPL). As LPL activity is regulated by insulin [20], preβ1-HDL production in poorly controlled diabetic patients may be impaired, especially in the postprandial state, as a result of insufficient insulin action. We examined whether the preβ1-HDL concentration changes postprandially in type 2 diabetic patients and if so, whether blood glucose (BG) control could affect this type of postprandial alterations.
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S. Hirayama et al. / Clinica Chimica Acta 401 (2009) 57–62 2.4. Immunoassay for preβ1-HDL
Table 1 Laboratory data for the diabetic patients and controls
Men/women Age (y) BMI (kg/m2) Systolic BP (mm Hg) Diastolic BP (mm Hg) FBG (mmol/l) HbA1c (%) TC (mmol/l) TG (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) ApoAI (g/l)
Diabetic patients (n = 20)
Controls (n = 9)
10/10 60.6 ± 13.0††† 25.7 ± 6.8† 130.8 ± 12.6†† 73.5 ± 11.0 10.4 ± 3.2††† 9.5 ± 2.6††† 5.22 ± 1.12 1.37 ± 0.69 3.31 ± 0.67†† 1.35 ± 0.41†† 1.26 ± 0.23†††
3/6 41.2 ± 12.5 21.1 ± 3.9 119.3 ± 7.9 68.0 ± 7.0 5.0 ± 0.4 5.1 ± 0.3 4.77 ± 0.85 0.93 ± 0.52 2.43 ± 0.46 1.87 ± 0.46 1.56 ± 0.23
Lipoprotein concentrations were measured before breakfast (in the fasting state). Values are presented as mean ± SD. †p b 0.05, ††p b 0.01, and †††p b 0.001, vs the controls. FBG, fasting blood glucose; HbA1c, hemoglobin A1c; TC, total cholesterol; TG, triglyceride; ApoAI, apolipoprotein AI.
We determined changes in the preβ1-HDL concentrations in type 2 diabetic patients, as well as in non-diabetic and normolipidemic subjects. 2. Materials and methods 2.1. Study subjects We selected 20 patients with type 2 diabetes who were admitted to our institute for treatment of their poor glycemic control. All patients satisfied the following criteria: fasting BG (FBG) N 7.0 mmol/l (126 mg/dl); postprandial BG (2 h after breakfast, 10:00 h) N 11.1 mmol/l (200 mg/dl); and hemoglobin A1c (HbA1c) N7.0%. We excluded patients who were taking hypolipidemic agents (typically statins and fibrates) and those with renal dysfunction (serum creatinine N2.0 mg/dl). After admission, we initiated strict BG control through a low-calorie diet and insulin therapy in 11 diabetic patients. Before initiation of insulin therapy, they stopped receiving anti-diabetic agents, if any. All of the study patients ate only diet meals provided by the hospital kitchen and stayed at hospital during the study. Their daily calorie intake was set at 25.0–27.5 kcal/kg/day, corresponding to 1200–1800 kcal/day (lipids 38–54 g; proteins 59–77 g; carbohydrates 150–250 g), and not changed until discharge. In addition, for 2 weeks, they underwent intensive insulin therapy with a combination of ultra-short-acting or short-acting insulin before each meal and intermediate-acting insulin at bedtime. As controls, nine healthy subjects were recruited with normal fasting lipid profiles [total cholesterol (TC) b 5.70 mmol/l (220 mg/dl), TG b1.70 mmol/l (150 mg/dl), HDL N1.04 mmol/l (40 mg/dl)], according to the criteria of the Japan Atherosclerosis Society [21]. The subjects ate usual meals three times/day, corresponding to an intake of approximately 2000 kcal/day (lipids 54 g; proteins 82 g; carbohydrates 290 g). They were only allowed to do lowlevel activities such as desk work. Informed consent was obtained from all subjects. The study protocol complied with the rules of the Helsinki Declaration. 2.2. Blood samples To examine postprandial changes in preβ1-HDL, we drew 7 blood samples/day, i.e., 30 min before and 2 h after meals (07:30, 10:00, 11:30, 14:00, 17:30, and 20:00 h) and at midnight. Postprandial changes were determined once in 9 controls and 20 diabetic patients, and twice in 11 diabetic patients: in the poor glycemic control (poor-GC) phase within a few days of admission, and in the improved glycemic control (imp-GC) phase after intensive treatment for 2 weeks, with fasting BG levels b8.3 mmol/l (150 mg/dl). For the measurement of preβ1-HDL, blood was immediately placed in a pre-cooled glass tube that contained EDTA-K2 (1 g/l), on ice [16,17]. Since the preβ1-HDL concentration steadily increases even at 0 °C after storage for 4 h [16], plasma was separated within 2 h by centrifugation at 0 °C, and diluted with 50% sucrose solution (1:21) for stabilization. The pretreated plasma was then frozen at −80 °C until assayed [16,17]. For the measurements of HbA1c and BG, blood samples were placed in glass tubes that contained NaF, to inhibit glucose catabolism. For measurements of other substances, the samples were placed in plain glass tubes, and serum was obtained and stored at 4 °C. 2.3. Measurement of lipid- and glucose-related variables Serum TC, TG, and BG concentrations were measured enzymatically, using an automated analyzer (Hitachi 7450; Hitachi, Japan). ApoAI concentrations were determined by a turbidimetric immunoassay, using a commercial kit (Sekisui Medical, Tokyo, Japan). HbA1c was determined by high-performance liquid chromatography. Albumin concentrations were measured by the bromocresol green method. Cholesteryl ester transfer protein (CETP) and lecithin:cholesterol acyltransferase (LCAT) masses were measured by enzyme-linked immunoassay [22]. The inter-assay and intra-assay coefficients of variation (CVs) were b 1% for TC, TG, BG, ApoAI, albumin, and HbA1c.
The preβ1-HDL concentration was quantified using a sandwich enzyme immunoassay (Sekisui Medical) [16,17]. All the pretreated samples from the same subjects were thawed simultaneously and applied to the same 96-well plate, which was precoated with specific monoclonal antibody for preβ1-HDL (mAb55201). Trapped lipoproteins were detected by a horseradish peroxidase-conjugated anti-ApoAI antibody with o-phenylenediamine and H2O2. The intra-assay and inter-assay CVs were b6%, as previously reported [16]. 2.5. Correction for postural changes in measured variables The concentrations of large intravascular components (e.g., lipoproteins and albumin) are dependent upon vascular tone, since fluid can move easily in or out of the blood vessels. Thus, lipoprotein concentrations can be modified by subject posture at blood sampling or by drugs that act on vascular tone. To determine authentic postprandial changes in the large intravascular components, we corrected the lipoprotein concentrations using simultaneously measured serum albumin concentrations [23]. Since albumin has a long half-life (19 days) [24], we assumed that the serum albumin concentration at any given time-point was the same as the concentration before breakfast. The correction was made using the following formula: [Lipoprotein]corrected = [Lipoprotein]point i × [Albumin]before breakfast ÷ [Albumin]point i [23]. In our previous study of 30 diabetic patients, uncorrected within-day variation in TC was twice as great as that corrected with albumin (10.9% vs 5.8%) [25]. In addition, this method revealed significant postprandial change which was masked in uncorrected data. We did not correct the blood glucose concentrations by this method. 2.6. Statistical analysis Data are presented as means ± SD. We conducted statistical analyses using commercial software StatMate III (Advanced Technology for Medicine & Science, Tokyo, Japan). The corresponding values for the same individuals were compared with the paired t-test, while the mean values of the different groups were compared by the Student's t-test and Welch's t-test. Differences were deemed to be statistically significant when the p value was b 0.05.
3. Results 3.1. Effects of intensive therapy on glucose and lipoprotein metabolism In the poor-GC phase, the mean FBG concentration was significantly higher in the diabetic patients than in the controls (Table 1). The TC and LDL-C concentrations were 9.4% and 36.2% higher in the poor-GC phase than in the controls. The fasting TG level was 47.3% higher in the poor-GC phase than in the controls (p = 0.10). The HDL-C and ApoAI levels were significantly lower (27.8% and 19.2%, respectively) in the poor-GC phase than in the controls. Intensive therapy with insulin and a low-calorie diet markedly improved BG control and the lipoprotein profiles in 11 diabetic patients. After treatment, the mean FBG and HbA1c values were reduced by 33.7% and 25.9%, respectively (Table 2). TC and LDL-C
Table 2 Laboratory data before and after intensive blood glucose control in diabetic patients Diabetic patients (n = 11) Men/women Age (y) BMI (kg/m2) Systolic BP (mm Hg) Diastolic BP (mm Hg) FBG (mmol/l) HbA1c (%) TC (mmol/l) TG (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) ApoAI (g/l)
Poor-GC phase
Imp-GC phase
5/6 63.5 ± 11.8††† 25.2 ± 4.3##,† 132.3 ± 13.6† 75.7 ± 13.3 10.4 ± 2.8##,††† 10.8 ± 2.7##,††† 5.51 ± 1.02## 1.60 ± 0.79#,† 3.29 ± 0.65##,† 1.33 ± 0.17†† 1.27 ± 0.11##,††
5/6 63.5 ± 11.8††† 24.5 ± 3.9 124.0 ± 13.1 73.0 ± 9.2 6.9 ± 0.9††† 8.0 ± 0.9††† 4.60 ± 0.63 1.13 ± 0.34 2.70 ± 0.52 1.25 ± 0.19††† 1.16 ± 0.11†††
Lipoprotein concentrations were measured before breakfast (in the fasting state). Values are presented as mean ± SD. #p b 0.05, ##p b 0.01, and ###p b 0.001, vs the imp-GC phase. †p b 0.05, ††p b 0.01, and †††p b 0.001, vs the controls in Table 1. poor-GC, poor glycemic control; imp-GC, improved glycemic control; FBG, fasting blood glucose; HbA1c, hemoglobin A1c; TC, total cholesterol; TG, triglyceride; LDL-C, low density lipoprotein-cholesterol; HDL-cholesterol; ApoAI, apolipoprotein AI.
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Fig. 1. Postprandial changes in the absolute preβ1-HDL (A), ApoAI (B), blood glucose (C) and TG (D) concentrations in diabetic patients and controls. Samples were obtained 30 min before and 2 h after each meal (07:30, 10:00, 11:30, 14:00, 17:30, and 20:00 h), and at midnight (24:00 h). Individual time-points correspond to Bbe (before breakfast), Baf (after breakfast), Lbe (before Lunch), Laf (after Lunch), Dbe (before dinner), Daf (after dinner), and Mid (midnight). Postprandial changes in the absolute preβ1-HDL levels in the poor-GC phase (n = 20) are shown by closed circles (●). ⁎p b 0.05, ⁎⁎p b 0.01, and ⁎⁎⁎p b 0.001, compared with individual fasting levels. †p b 0.05, ††p b 0.01, and †††p b 0.001, compared with corresponding values at the same sampling point of the controls (closed triangle (▲)). Differences that were not statistically significant are not indicated.
were decreased by 16.5% and 17.9%, respectively. The fasting TG levels decreased significantly by 29.4% in the imp-GC phase (p b 0.05), and were almost the same as those in the controls. The HDL-C
level in the imp-GC phase was as low as that in the poor-GC phase, whereas the level of ApoAI was significantly decreased in the imp-GC phase.
Fig. 2. Postprandial changes in the absolute preβ1-HDL (A), ApoAI (B), blood glucose (C) and TG (D) concentrations in the poor-GC and imp-GC phases of diabetic patients. Each sampling point and the statistical values (⁎ and †) are described in Fig. 1. Postprandial changes in the absolute preβ1-HDL levels in the poor-GC phase (n = 11) are shown by closed rhombus (♦). # p b 0.05, ## p b 0.01, and ### p b 0.001, compared with corresponding values at the same sampling point in the imp-GC phase (closed square (■)).
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Fig. 3. Postprandial changes in the CETP (A) and LCAT (B) concentrations in the poor-GC and imp-GC phases of diabetic patients and controls. Each sampling point and the statistical values (⁎, † and #) are described in Figs. 1 and 2.
3.2. Fasting preβ1-HDL concentration
3.4. Postprandial changes in absolute preβ1-HDL concentrations
The fasting preβ1-HDL concentration changed in parallel with the status of BG control in the diabetic patients; the mean absolute preβ1HDL concentration (expressed as the amount of ApoAI) was 28.3% higher in the poor-GC phase than in the controls (Fig. 1A). Intensive basal-bolus insulin therapy reduced the preβ1-HDL concentration by 28.4% in the imp-GC phase (Fig. 2A). The mean preβ1-HDL concentration in the imp-GC phase was as low as that in the controls (Figs. 1A, 2A). The mean relative preβ1-HDL concentration (expressed as the percentage of serum ApoAI) showed the same result (data not shown).
Unlike ApoAI, the postprandial changes in preβ1-HDL differed between the poor-GC and the imp-GC phases. In the poor-GC phase, preβ1-HDL was very high before breakfast, but was markedly reduced after breakfast (Figs. 1A, 2A). The preβ1-HDL level decreased gradually during the daytime (Fig. 1A, closed circle; Fig. 2A, closed rhombus). On the other hand, preβ1-HDL levels in the controls did not change significantly, and showed a significant reduction only after dinner (Fig. 1A, closed triangle). In the imp-GC phase, the preβ1-HDL levels were almost constant throughout the day (Fig. 2A, closed square). 3.5. Postprandial changes in both masses of CETP and LCAT
3.3. Postprandial changes in BG, TG, and ApoAI BG control greatly affected the postprandial pattern of TG, but did not change that of ApoAI (Fig. 2B, D). In the diabetic patients, the BG level markedly increased after each meal in the poor-GC phase (Figs. 1C, 2C). Although the BG levels were reduced at all sampling points in the imp-GC phase, they were still significantly increased in the postprandial states (after breakfast and dinner; Fig. 2C). At all sampling points, the TG levels were highest in the poor-GC phase of the diabetic patients (Figs. 1D, 2D). BG control reduced the TG levels by 16.8–37.2%, and the TG levels in the imp-GC phase were nearly the same as those in the controls. High TG levels were sustained for a longer time in the diabetic patients than in the controls, even in the imp-GC phase (⁎, compared with fasting levels in corresponding individuals; Figs. 1D, 2D). In both the controls and diabetic patients, the ApoAI levels showed no postprandial alterations but reached a minimum value at 24:00 h (Figs. 1B, 2B). The percentage reductions at 24:00 h were b5% (3.8% in the controls, 2.1% in the poor-GC phase, and 0.9% in the imp-GC phase, as compared to the individual fasting levels). Improvement in glycemic control did not significantly affect the postprandial pattern in ApoAI levels (Fig. 2B).
We measured the masses of CETP and LCAT, which are well-known regulatory factors in HDL metabolism. The CETP mass decreased gradually by 6.5–10.3% through the daytime, except at one point before dinner in the poor-GC phase, while those in the imp-GC phase and the controls showed almost no postprandial change in CETP mass (Fig. 3A). Although the baseline LCAT mass decreased after glycemic control, it did not exhibit any significant postprandial change in this study populations or change with the status of glycemic control (Fig. 3B). We calculated Δ preβ1-HDL and Δ CETP by subtracting the values in the imp-GC phase from those in the poor-GC phase. There was no significant correlation between them. However, the CETP/LCAT ratio tended to be correlated with the fasting preβ1-HDL concentration in the poor-GC phase (r = 0.40, p = 0.07). 4. Discussion The present study indicates that type 2 diabetic patients in the poor-GC phase have high preβ1-HDL levels only in the morning, followed by a gradual reduction until midnight, and that BG control diminishes this postprandial change. We found that the fasting preβ1-
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HDL concentration was 28.3% higher in the poor-GC phase than in the controls. However, the preβ1-HDL concentration declined to a similar level to the controls after breakfast and remained low until 24:00 h (Fig. 1A). Marked changes in the preβ1-HDL concentration ceased after 2 weeks of treatment with a low-calorie diet and intensive insulin therapy (Fig. 2A). The postprandial change of preβ1-HDL was very similar between the diabetic patients in the imp-GC phase and the controls. As seen in the poor-GC phase of the diabetic patients, the fasting preβ1-HDL concentration is elevated in atherosclerotic disorders and atherogenic conditions, such as coronary artery disease (CAD) [13], hemodialysis patients [14], hypercholesterolemia [11], and hypertriglyceridemia [12], but not altered regardless of sex and age [22]. Recently, it has been reported that smaller-sized HDL particles are increased in CAD [26,27]. The increase in preβ1-HDL may result from either delayed maturation of preβ1-HDL into α-migrating HDL [28] or activation of the ABCA1-pathway in atherosclerotic lesions [29]. The postprandial reduction in preβ1-HDL may be explained by a gradual reduction in CETP mass in the daytime (Fig. 3A). The preβ1HDL concentration increased in hypercholesterolemia with high CETP activity and was positively correlated with CETP mass in the subjects with normal triglyceride and HDL-C concentrations [11]. Since CETP promotes the conversion of α-migrating HDL into preβ1-HDL, low CETP activity might decrease preβ1-HDL generation in the postprandial state. Unfortunately, we failed to find a significant correlation between changes in preβ1-HDL (Δ preβ1-HDL) and those in CETP (Δ CETP) mass induced by intensive insulin therapy. In the poor-GC phase, however, the CETP/LCAT ratio tended to correlate with the fasting preβ1-HDL concentration. Since LCAT promotes the conversion of preβ1-HDL into α-migrating HDL, in the opposite direction of CETP [14], the balance of CETP and LCAT is very likely affects the preβ1-HDL concentration in the steady state. Huesca-Gómez et al. also reported that the CETP/LCAT ratio is a significant determinant of the HDL size distribution [30]. Brites et al. examined 14 male type 2 diabetic patients and evaluated the HDL subfractions using native 2D-gel electrophoresis [31]. Although they did not quantify the preβ1-HDL concentrations, they found not only preβ1-HDL, but also an additional preβ-migrating HDL (designated preβ0-HDL), which was slightly smaller than preβ1HDL. The estimated molecular weight was 40 kDa, while that of preβ1-HDL is 60–70 kDa. These findings suggest that preβ-HDL metabolism may be affected in type 2 diabetic patients. We cannot exclude the possibility that our antibody (mAb55201) might have higher reactivity with such smaller preβ-HDL. We hope to examine this in a future study. Another possible explanation for the postprandial reduction in preβ1-HDL is that preβ1-HDL seems to be adsorbed by TG-rich lipoprotein (TGRL) and released again as surface remnants, by lipoprotein lipase (LPL). Neary et al. administered 10% intralipid (fat emulsion) intravenously to six healthy subjects and measured the total preβ-HDL concentrations by crossed immunoelectrophoresis [32]. During the first 10 min, the total preβ1-HDL concentration decreased rapidly, and thereafter increased to the preinfusion level in all the subjects. The same immediate fall was detected when these researchers added intralipid to serum in vitro. They also carried out an in vitro experiment to examine the generation of preβ-HDL from TGRL using pre- and post-heparin plasma. Although the amount of HDL was the same, total preβ-HDL increased more in post-heparin than in preheparin plasma. This finding was particularly evident in hypertriglycemic patients following fat loading. Since heparin dissociates the binding of LPL to endothelial surfaces, these data might suggest that some preβ-HDL is derived from TGRL, being hydrolyzed by LPL. In the poor-GC phase, insufficient insulin action may attenuate LPL activity, leading to impaired release of preβ1-HDL from TGRL. The low LPL activity in type 2 diabetes and possible molecular mechanisms via protein kinase B activation by resistin and glucose-dependent
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insulinotropic polypeptide were already reported [33,34]. In the present study, a marked reduction in preβ1-HDL after breakfast was found only in the poor-GC phase of the diabetic patients (Figs. 1A, 2A). However, the postprandial changes in ApoAI were similar regardless of the study group or BG control (Figs. 1B, 2B). Postprandial hyperlipidemia was apparently worse in the poor-GC phase than in the imp-GC phase in the diabetic patients or controls (Figs. 1D, 2D). Thus, excess substrate (TGRL) in the poor-GC phase may place an excessive burden on reduced lipolytic activity, and increased TGRL may contribute to the reduction in preβ1-HDL concentration in the postprandial state. It seems likely that adsorption of preβ1-HDL is balanced by its release from TGRL after breakfast. There are 2 limitations to this study. First, the number of subjects was relatively small. Since the preβ1-HDL concentration increased significantly during storage, even at 0 °C, we had to separate and pretreat plasma samples within 2 h for stabilization. This experimental protocol made it difficult for us to enroll many healthy volunteers as study subjects. Second, we could not completely match the ages of diabetic patients and controls. However, this may not cause a serious problem because the preβ1-HDL concentration does not differ with age [22]. Further studies are required to elucidate the mechanism underlying the postprandial change in preβ1-HDL. In conclusion, the present study indicates that type 2 diabetic patients in the poor-GC phase have high preβ1-HDL levels only in the morning, with a gradual decline until midnight, and that BG control diminishes this postprandial change. Glucose metabolism may be involved in the modulation of preβ1-HDL metabolism in poorly controlled type 2 diabetic patients. Acknowledgements This research was partly supported by the Grants-in-Aid of Science Research from the Ministry of Education, Science, and Culture of Japan (No. 16590815, 2003-2004). We would like to thank Ms. Kaori Yoshida and Ms. Hiromi Ito for excellent technical assistance. References [1] Hirose T, Kawamori R. Diabetes in Japan. Curr Diab Rep 2005;5:226–9. [2] The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977–86. [3] Ohkubo Y, Kishikawa H, Araki E, et al. Intensive insulin therapy prevents the progression of diabetic microvascular complications in Japanese patients with non-insulin-dependent diabetes mellitus: a randomized prospective 6-year study. Diabetes Res Clin Pract 1995;28:103–7. [4] UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 1998;352:837–53. [5] Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med 1998;339:229–34. [6] Garcia MJ, McNamara PM, Gordon T, Kannel WB. Morbidity and mortality in diabetics in the Framingham population. Sixteen year follow-up study. Diabetes 1974;3:105–11. [7] Kannel WB, D'Agostino RB, Wilson PW, Belanger AJ, Gagnon DR. Lipoproteins, apolipoproteins, and low-density lipoprotein size among diabetics in the Framingham offspring study. Metabolism 1996;5:1267–72. [8] Krauss RM. Lipids and lipoproteins in patients with type 2 diabetes. Diabetes Care 2004;27:1496–504. [9] Hughes TA, Elam MB, Applegate WB, et al. Postprandial lipoprotein responses in hypertriglyceridemic subjects with and without cardiovascular disease. Metabolism 1995;44:1082–98. [10] Forrester JS, Makkar R, Shah PK. Increasing high-density lipoprotein cholesterol in dyslipidemia by cholesteryl ester transfer protein inhibition: an update for clinicians. Circulation 2005;111:1847–54. [11] Miida T, Ozaki K, Murakami T, et al. Prebeta1-high-density lipoprotein (prebeta1HDL) concentration can change with low-density lipoprotein-cholesterol (LDL-C) concentration independent of cholesteryl ester transfer protein (CETP). Clin Chim Acta 2000;292:69–80. [12] Miida T, Sakai K, Ozaki K, et al. Bezafibrate increases preβ1-HDL at the expense of HDL2b in hypertriglyceridemia. Arterioscler Thromb Vasc Biol 2000;20:2428–33. [13] Miida T, Nakamura Y, Inano K, et al. Preβ1-high-density lipoprotein increases in coronary artery disease. Clin Chem 1996;42:1992–5.
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[14] Miida T, Miyazaki O, Hanyu O, et al. LCAT-dependent conversion of preβ1-HDL into α-migrating HDL is severely delayed in hemodialysis patients. J Am Soc Nephrol 2003;14:732–8. [15] Miida T, Kawano M, Fielding PE, Fielding CJ. Regulation of the concentration of prebeta high density lipoprotein in human plasma by cell membrane and lecithin: cholesterol acyltransferase activity. Biochemistry 1992;1:11112–7. [16] Miida T, Miyazaki O, Nakamura Y, et al. Analytical performance of a sandwich enzyme immunoassay for preβ1-HDL in stabilized plasma. J Lipid Res 2003;4:645–50. [17] Hirayama S, Miida T, Miyazaki O, Aizawa Y. Preβ1-HDL concentration is a predictor of carotid atherosclerosis in type 2 diabetic patients. Diabetes Care 2007;30:1289–91. [18] Barrans A, Collet X, Barbaras R, et al. Hepatic lipase induces the formation of preβ1 high density lipoprotein (HDL) from triglycerol-rich HDL2: a study comparing liver perfusion to in vitro incubation with lipases. J Biol Chem 1994;269:11572–7. [19] Guendouzi K, Jaspard B, Barbaras R, et al. Biochemical and physical properties of remnant-HDL2 and of pre-β1-HDL produced by hepatic lipase. Biochemistry 1999;38:2762–8. [20] Ong JM, Kirchgessner TG, Schotz MC, Kern PA. Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes. J Biol Chem 1988;263:12933–8. [21] Hata Y, Mabuchi H, Saito Y, et al. Working committee on JAS guideline for diagnosis and treatment of hyperlipidemias. Report of the Japan Atherosclerosis Society (JAS) guideline for diagnosis and treatment of hyperlipidemia in Japanese adults. J Atheroscler Thromb 2002;9:1–27. [22] Miida T, Obayashi K, Seino U, et al. LCAT-dependent conversion rate is a determinant of plasma prebeta1-HDL concentration in healthy Japanese. Clin Chim Acta 2004;350:107–14. [23] Miida T, Nakamura Y, Mezaki T, et al. LDL-cholesterol and HDL-cholesterol decrease during the day. Ann Clin Biochem 2002;39:241–9. [24] Peters Jr T. Serum albumin. Adv Clin Chem 1970;13:37–111. [25] Miida T, Sasaki H, Sato K, et al. Postural change and within-day variation in total cholesterol and high-density lipoprotein-cholesterol levels. Rinsyo Byori 1996;44:860–4.
[26] Suzuki M, Wada H, Maeda S, et al. Increased plasma lipid-poor apolipoprotein A-I in patients with coronary artery disease. Clin Chem 2004;51:132–7. [27] Okazaki M, Usui S, Fukui A, Kubota I, Tomoike H. Component analysis of HPLC profiles of unique lipoprotein subclass cholesterols for detection of coronary artery disease. Clin Chem 2006;52:2049–53. [28] Chetiveaux M, Lalanne F, Lambert G, Zair Y, Ouguerram K, Krempf M. Kinetics of prebeta1 HDL and alphaHDL in type II diabetic patients. Eur J Clin Investig 2006;36:29–34. [29] Joyce CW, Amar MJ, Lambert G, et al. The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice. Proc Natl Acad Sci U S A 2002;99:407–12. [30] Huesca-Gómez C, Carreón-Torres E, Nepomuceno-Mejía T, et al. Contribution of cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase to HDL size distribution. Endocr Res 2004;30:403–15. [31] Brites FD, Bonavita CD, De Geitere C, et al. Abnormal capacity to induce cholesterol efflux and a new LpA-I pre-beta particle in type 2 diabetic patients. Clin Chim Acta 1999;279:1–14. [32] Neary RH, Bhatnagar D, Durrington P, Ishola M, Arrol S, Mackness M. An investigation of the role of lecithin:cholesterol acyltransferase and triglyceriderich lipoproteins in the metabolism of pre-beta high density lipoproteins. Atherosclerosis 1991;89:135–48. [33] Annuzzi G, Giacco R, Patti L, et al. Postprandial chylomicrons and adipose tissue lipoprotein lipase are altered in type 2 diabetes independently of obesity and whole-body insulin resistance. Nutr Metab Cardiovasc Dis 2008;18:531–8. [34] Kim SJ, Nian C, McIntosh CH. Resistin is a key mediator of glucose-dependent insulinotropic polypeptide (GIP) stimulation of lipoprotein lipase (LPL) activity in adipocytes. J Biol Chem 2007;282:34139–47.
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n c h i m
Preβ1-HDL is elevated in the fasting state, but markedly reduced postprandially in poorly controlled type 2 diabetic patients Satoshi Hirayama a, Takako Ito b, Osamu Miyazaki c, Takashi Kamimura b, Osamu Hanyu b, Utako Seino d, Seiki Ito b, Yoshifusa Aizawa b, Takashi Miida a,⁎ a
Department of Clinical Laboratory Medicine, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan Division of Endocrinology and Metabolism, Niigata University, Niigata, Japan Sekisui Medical, Tsukuba Research Institute, Ibaraki, Japan d Division of Clinical Laboratory Medicine, Niigata University, Niigata, Japan b c
a r t i c l e
i n f o
Article history: Received 7 March 2008 Received in revised form 26 October 2008 Accepted 7 November 2008 Available online 17 November 2008 Keywords: Prebeta-HDL Postprandial change CETP LCAT
a b s t r a c t Background: Preβ1-HDL is a minor HDL subfraction that is an initial acceptor of cellular free cholesterol. Preβ1-HDL is elevated in hypertriglyceridemia, which is exaggerated with postprandial hyperglycemia. We investigated whether the preβ1-HDL concentration changes postprandially in type 2 diabetic patients and blood glucose (BG) control reduces this change. Methods: We examined 9 healthy controls and 20 diabetic patients with poor BG control. Seven blood samples (30 min before and 2 h after each meal, and at midnight) were obtained daily in the poor (poor-GC: n = 20) and improved (imp-GC: n = 11) glycemic control phases of diabetic patients after intensive insulin therapy and a low-calorie diet. Results: The preβ1-HDL concentration did not change postprandially in the controls. However, the fasting preβ1-HDL concentration in the poor-GC phase was 28.3% higher than in the controls (25.4 ± 6.8 vs 19.8 ± 6.9 mg/l ApoAI, p b 0.05) and decreased markedly after breakfast (20.9 ± 7.7 mg/l ApoAI, p b 0.01). In the impGC phase, the preβ1-HDL concentration showed no morning surge, as in the controls. Conclusions: Type 2 diabetic patients in the poor-GC phase have high preβ1-HDL levels in the morning, followed by a gradual reduction until midnight. BG control diminishes this postprandial change. Glucose metabolism may be involved in modulating reverse cholesterol transport in type 2 diabetic patients. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The incidence of type 2 diabetes has increased in Japan, as the lifestyle has been somewhat ‘Westernized’ over the last 60 y [1]. Although strict glycemic control reduces diabetic microangiopathy [2,3], it does not significantly reduce cardiovascular disease (CVD) [4], which remains the leading cause of death in diabetic patients [5,6]. Diabetic patients often have high triglyceride (TG) and low HDL-cholesterol (HDL-C) levels [7]. Patients with such lipoprotein profiles are at higher risk of CVD than normolipidemic diabetics [5,8]. Furthermore, hypertriglyceridemia is often exaggerated in the postprandial state, which is another independent risk factor for CVD [9].
Abbreviations: CVD, Cardiovascular disease; TG, Triglyceride; HDL-C, HDL-cholesterol; ApoAI, Apolipoprotein AI; TGRL, Triglyceride rich lipoprotein; LPL, Lipoprotein lipase; CETP, Cholesteryl ester transfer protein; LCAT, Lecithin cholesterol acyltransferase. ⁎ Corresponding author. Tel.: +81 3 5802 1104; fax: +81 3 5684 1609. E-mail address: [email protected] (T. Miida). 0009-8981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.11.010
HDL removes excess cholesterol from peripheral tissues and transports it to the liver for excretion via the bile (reverse cholesterol transport) [10]. Since HDL particles vary in size, composition, and function, they are classified into several subfractions by different procedures [11–17]. Preβ1-HDL is a minor apolipoprotein AI (ApoAI)containing subfraction, which was first identified by native twodimensional gel (2D-gel) electrophoresis. Preβ1-HDL is primarily composed of ApoAI and phospholipids (surface components of lipoproteins) and it has little core lipid. Experimental data indicate that preβ1-HDL serves as an initial acceptor of cellular cholesterol [15]. The fasting preβ1-HDL concentration is elevated in hypertriglyceridemia [12]. This may reflect enhanced production of preβ1-HDL from αmigrating, TG-rich HDL particles by hepatic lipase [18,19], or the surface remnants of TG-rich lipoproteins (TGRL) hydrolyzed by lipoprotein lipase (LPL). As LPL activity is regulated by insulin [20], preβ1-HDL production in poorly controlled diabetic patients may be impaired, especially in the postprandial state, as a result of insufficient insulin action. We examined whether the preβ1-HDL concentration changes postprandially in type 2 diabetic patients and if so, whether blood glucose (BG) control could affect this type of postprandial alterations.
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Table 1 Laboratory data for the diabetic patients and controls
Men/women Age (y) BMI (kg/m2) Systolic BP (mm Hg) Diastolic BP (mm Hg) FBG (mmol/l) HbA1c (%) TC (mmol/l) TG (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) ApoAI (g/l)
Diabetic patients (n = 20)
Controls (n = 9)
10/10 60.6 ± 13.0††† 25.7 ± 6.8† 130.8 ± 12.6†† 73.5 ± 11.0 10.4 ± 3.2††† 9.5 ± 2.6††† 5.22 ± 1.12 1.37 ± 0.69 3.31 ± 0.67†† 1.35 ± 0.41†† 1.26 ± 0.23†††
3/6 41.2 ± 12.5 21.1 ± 3.9 119.3 ± 7.9 68.0 ± 7.0 5.0 ± 0.4 5.1 ± 0.3 4.77 ± 0.85 0.93 ± 0.52 2.43 ± 0.46 1.87 ± 0.46 1.56 ± 0.23
Lipoprotein concentrations were measured before breakfast (in the fasting state). Values are presented as mean ± SD. †p b 0.05, ††p b 0.01, and †††p b 0.001, vs the controls. FBG, fasting blood glucose; HbA1c, hemoglobin A1c; TC, total cholesterol; TG, triglyceride; ApoAI, apolipoprotein AI.
We determined changes in the preβ1-HDL concentrations in type 2 diabetic patients, as well as in non-diabetic and normolipidemic subjects. 2. Materials and methods 2.1. Study subjects We selected 20 patients with type 2 diabetes who were admitted to our institute for treatment of their poor glycemic control. All patients satisfied the following criteria: fasting BG (FBG) N 7.0 mmol/l (126 mg/dl); postprandial BG (2 h after breakfast, 10:00 h) N 11.1 mmol/l (200 mg/dl); and hemoglobin A1c (HbA1c) N7.0%. We excluded patients who were taking hypolipidemic agents (typically statins and fibrates) and those with renal dysfunction (serum creatinine N2.0 mg/dl). After admission, we initiated strict BG control through a low-calorie diet and insulin therapy in 11 diabetic patients. Before initiation of insulin therapy, they stopped receiving anti-diabetic agents, if any. All of the study patients ate only diet meals provided by the hospital kitchen and stayed at hospital during the study. Their daily calorie intake was set at 25.0–27.5 kcal/kg/day, corresponding to 1200–1800 kcal/day (lipids 38–54 g; proteins 59–77 g; carbohydrates 150–250 g), and not changed until discharge. In addition, for 2 weeks, they underwent intensive insulin therapy with a combination of ultra-short-acting or short-acting insulin before each meal and intermediate-acting insulin at bedtime. As controls, nine healthy subjects were recruited with normal fasting lipid profiles [total cholesterol (TC) b 5.70 mmol/l (220 mg/dl), TG b1.70 mmol/l (150 mg/dl), HDL N1.04 mmol/l (40 mg/dl)], according to the criteria of the Japan Atherosclerosis Society [21]. The subjects ate usual meals three times/day, corresponding to an intake of approximately 2000 kcal/day (lipids 54 g; proteins 82 g; carbohydrates 290 g). They were only allowed to do lowlevel activities such as desk work. Informed consent was obtained from all subjects. The study protocol complied with the rules of the Helsinki Declaration. 2.2. Blood samples To examine postprandial changes in preβ1-HDL, we drew 7 blood samples/day, i.e., 30 min before and 2 h after meals (07:30, 10:00, 11:30, 14:00, 17:30, and 20:00 h) and at midnight. Postprandial changes were determined once in 9 controls and 20 diabetic patients, and twice in 11 diabetic patients: in the poor glycemic control (poor-GC) phase within a few days of admission, and in the improved glycemic control (imp-GC) phase after intensive treatment for 2 weeks, with fasting BG levels b8.3 mmol/l (150 mg/dl). For the measurement of preβ1-HDL, blood was immediately placed in a pre-cooled glass tube that contained EDTA-K2 (1 g/l), on ice [16,17]. Since the preβ1-HDL concentration steadily increases even at 0 °C after storage for 4 h [16], plasma was separated within 2 h by centrifugation at 0 °C, and diluted with 50% sucrose solution (1:21) for stabilization. The pretreated plasma was then frozen at −80 °C until assayed [16,17]. For the measurements of HbA1c and BG, blood samples were placed in glass tubes that contained NaF, to inhibit glucose catabolism. For measurements of other substances, the samples were placed in plain glass tubes, and serum was obtained and stored at 4 °C. 2.3. Measurement of lipid- and glucose-related variables Serum TC, TG, and BG concentrations were measured enzymatically, using an automated analyzer (Hitachi 7450; Hitachi, Japan). ApoAI concentrations were determined by a turbidimetric immunoassay, using a commercial kit (Sekisui Medical, Tokyo, Japan). HbA1c was determined by high-performance liquid chromatography. Albumin concentrations were measured by the bromocresol green method. Cholesteryl ester transfer protein (CETP) and lecithin:cholesterol acyltransferase (LCAT) masses were measured by enzyme-linked immunoassay [22]. The inter-assay and intra-assay coefficients of variation (CVs) were b 1% for TC, TG, BG, ApoAI, albumin, and HbA1c.
The preβ1-HDL concentration was quantified using a sandwich enzyme immunoassay (Sekisui Medical) [16,17]. All the pretreated samples from the same subjects were thawed simultaneously and applied to the same 96-well plate, which was precoated with specific monoclonal antibody for preβ1-HDL (mAb55201). Trapped lipoproteins were detected by a horseradish peroxidase-conjugated anti-ApoAI antibody with o-phenylenediamine and H2O2. The intra-assay and inter-assay CVs were b6%, as previously reported [16]. 2.5. Correction for postural changes in measured variables The concentrations of large intravascular components (e.g., lipoproteins and albumin) are dependent upon vascular tone, since fluid can move easily in or out of the blood vessels. Thus, lipoprotein concentrations can be modified by subject posture at blood sampling or by drugs that act on vascular tone. To determine authentic postprandial changes in the large intravascular components, we corrected the lipoprotein concentrations using simultaneously measured serum albumin concentrations [23]. Since albumin has a long half-life (19 days) [24], we assumed that the serum albumin concentration at any given time-point was the same as the concentration before breakfast. The correction was made using the following formula: [Lipoprotein]corrected = [Lipoprotein]point i × [Albumin]before breakfast ÷ [Albumin]point i [23]. In our previous study of 30 diabetic patients, uncorrected within-day variation in TC was twice as great as that corrected with albumin (10.9% vs 5.8%) [25]. In addition, this method revealed significant postprandial change which was masked in uncorrected data. We did not correct the blood glucose concentrations by this method. 2.6. Statistical analysis Data are presented as means ± SD. We conducted statistical analyses using commercial software StatMate III (Advanced Technology for Medicine & Science, Tokyo, Japan). The corresponding values for the same individuals were compared with the paired t-test, while the mean values of the different groups were compared by the Student's t-test and Welch's t-test. Differences were deemed to be statistically significant when the p value was b 0.05.
3. Results 3.1. Effects of intensive therapy on glucose and lipoprotein metabolism In the poor-GC phase, the mean FBG concentration was significantly higher in the diabetic patients than in the controls (Table 1). The TC and LDL-C concentrations were 9.4% and 36.2% higher in the poor-GC phase than in the controls. The fasting TG level was 47.3% higher in the poor-GC phase than in the controls (p = 0.10). The HDL-C and ApoAI levels were significantly lower (27.8% and 19.2%, respectively) in the poor-GC phase than in the controls. Intensive therapy with insulin and a low-calorie diet markedly improved BG control and the lipoprotein profiles in 11 diabetic patients. After treatment, the mean FBG and HbA1c values were reduced by 33.7% and 25.9%, respectively (Table 2). TC and LDL-C
Table 2 Laboratory data before and after intensive blood glucose control in diabetic patients Diabetic patients (n = 11) Men/women Age (y) BMI (kg/m2) Systolic BP (mm Hg) Diastolic BP (mm Hg) FBG (mmol/l) HbA1c (%) TC (mmol/l) TG (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) ApoAI (g/l)
Poor-GC phase
Imp-GC phase
5/6 63.5 ± 11.8††† 25.2 ± 4.3##,† 132.3 ± 13.6† 75.7 ± 13.3 10.4 ± 2.8##,††† 10.8 ± 2.7##,††† 5.51 ± 1.02## 1.60 ± 0.79#,† 3.29 ± 0.65##,† 1.33 ± 0.17†† 1.27 ± 0.11##,††
5/6 63.5 ± 11.8††† 24.5 ± 3.9 124.0 ± 13.1 73.0 ± 9.2 6.9 ± 0.9††† 8.0 ± 0.9††† 4.60 ± 0.63 1.13 ± 0.34 2.70 ± 0.52 1.25 ± 0.19††† 1.16 ± 0.11†††
Lipoprotein concentrations were measured before breakfast (in the fasting state). Values are presented as mean ± SD. #p b 0.05, ##p b 0.01, and ###p b 0.001, vs the imp-GC phase. †p b 0.05, ††p b 0.01, and †††p b 0.001, vs the controls in Table 1. poor-GC, poor glycemic control; imp-GC, improved glycemic control; FBG, fasting blood glucose; HbA1c, hemoglobin A1c; TC, total cholesterol; TG, triglyceride; LDL-C, low density lipoprotein-cholesterol; HDL-cholesterol; ApoAI, apolipoprotein AI.
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Fig. 1. Postprandial changes in the absolute preβ1-HDL (A), ApoAI (B), blood glucose (C) and TG (D) concentrations in diabetic patients and controls. Samples were obtained 30 min before and 2 h after each meal (07:30, 10:00, 11:30, 14:00, 17:30, and 20:00 h), and at midnight (24:00 h). Individual time-points correspond to Bbe (before breakfast), Baf (after breakfast), Lbe (before Lunch), Laf (after Lunch), Dbe (before dinner), Daf (after dinner), and Mid (midnight). Postprandial changes in the absolute preβ1-HDL levels in the poor-GC phase (n = 20) are shown by closed circles (●). ⁎p b 0.05, ⁎⁎p b 0.01, and ⁎⁎⁎p b 0.001, compared with individual fasting levels. †p b 0.05, ††p b 0.01, and †††p b 0.001, compared with corresponding values at the same sampling point of the controls (closed triangle (▲)). Differences that were not statistically significant are not indicated.
were decreased by 16.5% and 17.9%, respectively. The fasting TG levels decreased significantly by 29.4% in the imp-GC phase (p b 0.05), and were almost the same as those in the controls. The HDL-C
level in the imp-GC phase was as low as that in the poor-GC phase, whereas the level of ApoAI was significantly decreased in the imp-GC phase.
Fig. 2. Postprandial changes in the absolute preβ1-HDL (A), ApoAI (B), blood glucose (C) and TG (D) concentrations in the poor-GC and imp-GC phases of diabetic patients. Each sampling point and the statistical values (⁎ and †) are described in Fig. 1. Postprandial changes in the absolute preβ1-HDL levels in the poor-GC phase (n = 11) are shown by closed rhombus (♦). # p b 0.05, ## p b 0.01, and ### p b 0.001, compared with corresponding values at the same sampling point in the imp-GC phase (closed square (■)).
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Fig. 3. Postprandial changes in the CETP (A) and LCAT (B) concentrations in the poor-GC and imp-GC phases of diabetic patients and controls. Each sampling point and the statistical values (⁎, † and #) are described in Figs. 1 and 2.
3.2. Fasting preβ1-HDL concentration
3.4. Postprandial changes in absolute preβ1-HDL concentrations
The fasting preβ1-HDL concentration changed in parallel with the status of BG control in the diabetic patients; the mean absolute preβ1HDL concentration (expressed as the amount of ApoAI) was 28.3% higher in the poor-GC phase than in the controls (Fig. 1A). Intensive basal-bolus insulin therapy reduced the preβ1-HDL concentration by 28.4% in the imp-GC phase (Fig. 2A). The mean preβ1-HDL concentration in the imp-GC phase was as low as that in the controls (Figs. 1A, 2A). The mean relative preβ1-HDL concentration (expressed as the percentage of serum ApoAI) showed the same result (data not shown).
Unlike ApoAI, the postprandial changes in preβ1-HDL differed between the poor-GC and the imp-GC phases. In the poor-GC phase, preβ1-HDL was very high before breakfast, but was markedly reduced after breakfast (Figs. 1A, 2A). The preβ1-HDL level decreased gradually during the daytime (Fig. 1A, closed circle; Fig. 2A, closed rhombus). On the other hand, preβ1-HDL levels in the controls did not change significantly, and showed a significant reduction only after dinner (Fig. 1A, closed triangle). In the imp-GC phase, the preβ1-HDL levels were almost constant throughout the day (Fig. 2A, closed square). 3.5. Postprandial changes in both masses of CETP and LCAT
3.3. Postprandial changes in BG, TG, and ApoAI BG control greatly affected the postprandial pattern of TG, but did not change that of ApoAI (Fig. 2B, D). In the diabetic patients, the BG level markedly increased after each meal in the poor-GC phase (Figs. 1C, 2C). Although the BG levels were reduced at all sampling points in the imp-GC phase, they were still significantly increased in the postprandial states (after breakfast and dinner; Fig. 2C). At all sampling points, the TG levels were highest in the poor-GC phase of the diabetic patients (Figs. 1D, 2D). BG control reduced the TG levels by 16.8–37.2%, and the TG levels in the imp-GC phase were nearly the same as those in the controls. High TG levels were sustained for a longer time in the diabetic patients than in the controls, even in the imp-GC phase (⁎, compared with fasting levels in corresponding individuals; Figs. 1D, 2D). In both the controls and diabetic patients, the ApoAI levels showed no postprandial alterations but reached a minimum value at 24:00 h (Figs. 1B, 2B). The percentage reductions at 24:00 h were b5% (3.8% in the controls, 2.1% in the poor-GC phase, and 0.9% in the imp-GC phase, as compared to the individual fasting levels). Improvement in glycemic control did not significantly affect the postprandial pattern in ApoAI levels (Fig. 2B).
We measured the masses of CETP and LCAT, which are well-known regulatory factors in HDL metabolism. The CETP mass decreased gradually by 6.5–10.3% through the daytime, except at one point before dinner in the poor-GC phase, while those in the imp-GC phase and the controls showed almost no postprandial change in CETP mass (Fig. 3A). Although the baseline LCAT mass decreased after glycemic control, it did not exhibit any significant postprandial change in this study populations or change with the status of glycemic control (Fig. 3B). We calculated Δ preβ1-HDL and Δ CETP by subtracting the values in the imp-GC phase from those in the poor-GC phase. There was no significant correlation between them. However, the CETP/LCAT ratio tended to be correlated with the fasting preβ1-HDL concentration in the poor-GC phase (r = 0.40, p = 0.07). 4. Discussion The present study indicates that type 2 diabetic patients in the poor-GC phase have high preβ1-HDL levels only in the morning, followed by a gradual reduction until midnight, and that BG control diminishes this postprandial change. We found that the fasting preβ1-
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HDL concentration was 28.3% higher in the poor-GC phase than in the controls. However, the preβ1-HDL concentration declined to a similar level to the controls after breakfast and remained low until 24:00 h (Fig. 1A). Marked changes in the preβ1-HDL concentration ceased after 2 weeks of treatment with a low-calorie diet and intensive insulin therapy (Fig. 2A). The postprandial change of preβ1-HDL was very similar between the diabetic patients in the imp-GC phase and the controls. As seen in the poor-GC phase of the diabetic patients, the fasting preβ1-HDL concentration is elevated in atherosclerotic disorders and atherogenic conditions, such as coronary artery disease (CAD) [13], hemodialysis patients [14], hypercholesterolemia [11], and hypertriglyceridemia [12], but not altered regardless of sex and age [22]. Recently, it has been reported that smaller-sized HDL particles are increased in CAD [26,27]. The increase in preβ1-HDL may result from either delayed maturation of preβ1-HDL into α-migrating HDL [28] or activation of the ABCA1-pathway in atherosclerotic lesions [29]. The postprandial reduction in preβ1-HDL may be explained by a gradual reduction in CETP mass in the daytime (Fig. 3A). The preβ1HDL concentration increased in hypercholesterolemia with high CETP activity and was positively correlated with CETP mass in the subjects with normal triglyceride and HDL-C concentrations [11]. Since CETP promotes the conversion of α-migrating HDL into preβ1-HDL, low CETP activity might decrease preβ1-HDL generation in the postprandial state. Unfortunately, we failed to find a significant correlation between changes in preβ1-HDL (Δ preβ1-HDL) and those in CETP (Δ CETP) mass induced by intensive insulin therapy. In the poor-GC phase, however, the CETP/LCAT ratio tended to correlate with the fasting preβ1-HDL concentration. Since LCAT promotes the conversion of preβ1-HDL into α-migrating HDL, in the opposite direction of CETP [14], the balance of CETP and LCAT is very likely affects the preβ1-HDL concentration in the steady state. Huesca-Gómez et al. also reported that the CETP/LCAT ratio is a significant determinant of the HDL size distribution [30]. Brites et al. examined 14 male type 2 diabetic patients and evaluated the HDL subfractions using native 2D-gel electrophoresis [31]. Although they did not quantify the preβ1-HDL concentrations, they found not only preβ1-HDL, but also an additional preβ-migrating HDL (designated preβ0-HDL), which was slightly smaller than preβ1HDL. The estimated molecular weight was 40 kDa, while that of preβ1-HDL is 60–70 kDa. These findings suggest that preβ-HDL metabolism may be affected in type 2 diabetic patients. We cannot exclude the possibility that our antibody (mAb55201) might have higher reactivity with such smaller preβ-HDL. We hope to examine this in a future study. Another possible explanation for the postprandial reduction in preβ1-HDL is that preβ1-HDL seems to be adsorbed by TG-rich lipoprotein (TGRL) and released again as surface remnants, by lipoprotein lipase (LPL). Neary et al. administered 10% intralipid (fat emulsion) intravenously to six healthy subjects and measured the total preβ-HDL concentrations by crossed immunoelectrophoresis [32]. During the first 10 min, the total preβ1-HDL concentration decreased rapidly, and thereafter increased to the preinfusion level in all the subjects. The same immediate fall was detected when these researchers added intralipid to serum in vitro. They also carried out an in vitro experiment to examine the generation of preβ-HDL from TGRL using pre- and post-heparin plasma. Although the amount of HDL was the same, total preβ-HDL increased more in post-heparin than in preheparin plasma. This finding was particularly evident in hypertriglycemic patients following fat loading. Since heparin dissociates the binding of LPL to endothelial surfaces, these data might suggest that some preβ-HDL is derived from TGRL, being hydrolyzed by LPL. In the poor-GC phase, insufficient insulin action may attenuate LPL activity, leading to impaired release of preβ1-HDL from TGRL. The low LPL activity in type 2 diabetes and possible molecular mechanisms via protein kinase B activation by resistin and glucose-dependent
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insulinotropic polypeptide were already reported [33,34]. In the present study, a marked reduction in preβ1-HDL after breakfast was found only in the poor-GC phase of the diabetic patients (Figs. 1A, 2A). However, the postprandial changes in ApoAI were similar regardless of the study group or BG control (Figs. 1B, 2B). Postprandial hyperlipidemia was apparently worse in the poor-GC phase than in the imp-GC phase in the diabetic patients or controls (Figs. 1D, 2D). Thus, excess substrate (TGRL) in the poor-GC phase may place an excessive burden on reduced lipolytic activity, and increased TGRL may contribute to the reduction in preβ1-HDL concentration in the postprandial state. It seems likely that adsorption of preβ1-HDL is balanced by its release from TGRL after breakfast. There are 2 limitations to this study. First, the number of subjects was relatively small. Since the preβ1-HDL concentration increased significantly during storage, even at 0 °C, we had to separate and pretreat plasma samples within 2 h for stabilization. This experimental protocol made it difficult for us to enroll many healthy volunteers as study subjects. Second, we could not completely match the ages of diabetic patients and controls. However, this may not cause a serious problem because the preβ1-HDL concentration does not differ with age [22]. Further studies are required to elucidate the mechanism underlying the postprandial change in preβ1-HDL. In conclusion, the present study indicates that type 2 diabetic patients in the poor-GC phase have high preβ1-HDL levels only in the morning, with a gradual decline until midnight, and that BG control diminishes this postprandial change. Glucose metabolism may be involved in the modulation of preβ1-HDL metabolism in poorly controlled type 2 diabetic patients. Acknowledgements This research was partly supported by the Grants-in-Aid of Science Research from the Ministry of Education, Science, and Culture of Japan (No. 16590815, 2003-2004). We would like to thank Ms. Kaori Yoshida and Ms. Hiromi Ito for excellent technical assistance. References [1] Hirose T, Kawamori R. Diabetes in Japan. Curr Diab Rep 2005;5:226–9. [2] The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977–86. [3] Ohkubo Y, Kishikawa H, Araki E, et al. Intensive insulin therapy prevents the progression of diabetic microvascular complications in Japanese patients with non-insulin-dependent diabetes mellitus: a randomized prospective 6-year study. Diabetes Res Clin Pract 1995;28:103–7. [4] UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 1998;352:837–53. [5] Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med 1998;339:229–34. [6] Garcia MJ, McNamara PM, Gordon T, Kannel WB. Morbidity and mortality in diabetics in the Framingham population. Sixteen year follow-up study. Diabetes 1974;3:105–11. [7] Kannel WB, D'Agostino RB, Wilson PW, Belanger AJ, Gagnon DR. Lipoproteins, apolipoproteins, and low-density lipoprotein size among diabetics in the Framingham offspring study. Metabolism 1996;5:1267–72. [8] Krauss RM. Lipids and lipoproteins in patients with type 2 diabetes. Diabetes Care 2004;27:1496–504. [9] Hughes TA, Elam MB, Applegate WB, et al. Postprandial lipoprotein responses in hypertriglyceridemic subjects with and without cardiovascular disease. Metabolism 1995;44:1082–98. [10] Forrester JS, Makkar R, Shah PK. Increasing high-density lipoprotein cholesterol in dyslipidemia by cholesteryl ester transfer protein inhibition: an update for clinicians. Circulation 2005;111:1847–54. [11] Miida T, Ozaki K, Murakami T, et al. Prebeta1-high-density lipoprotein (prebeta1HDL) concentration can change with low-density lipoprotein-cholesterol (LDL-C) concentration independent of cholesteryl ester transfer protein (CETP). Clin Chim Acta 2000;292:69–80. [12] Miida T, Sakai K, Ozaki K, et al. Bezafibrate increases preβ1-HDL at the expense of HDL2b in hypertriglyceridemia. Arterioscler Thromb Vasc Biol 2000;20:2428–33. [13] Miida T, Nakamura Y, Inano K, et al. Preβ1-high-density lipoprotein increases in coronary artery disease. Clin Chem 1996;42:1992–5.
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