Atherosclerosis 231 (2013) 405e410
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Impact of serum amyloid A on cellular cholesterol efflux to serum in type 2 diabetes mellitus J.G.S. Tsun, S.W.M. Shiu, Y. Wong, S. Yung, T.M. Chan, K.C.B. Tan* Department of Medicine, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 May 2013 Received in revised form 7 September 2013 Accepted 10 October 2013 Available online 18 October 2013
Objective: Serum amyloid A (SAA) is an acute phase response protein and has apolipoprotein properties. Since type 2 diabetes is associated with chronic subclinical inflammation, the objective of this study is to investigate the changes in SAA level in type 2 diabetic patients and to evaluate the relationship between SAA and the capacity of serum to induce cellular cholesterol efflux via the two known cholesterol transporters, scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter G1 (ABCG1). Methods: 264 patients with type 2 diabetes mellitus (42% with normoalbuminuria, 30% microalbuminuria, and 28% proteinuria) and 275 non-diabetic controls were recruited. SAA was measured by ELISA. SR-BI and ABCG1-mediated cholesterol efflux to serum were determined by measuring the transfer of [3H]cholesterol from Fu5AH rat hepatoma cells expressing SR-BI and from human ABCG1transfected CHO-K1 cells to the medium containing the tested serum respectively. Results: SAA was significantly increased in diabetic patients with incipient or overt nephropathy. Both SR-BI and ABCG1-mediated cholesterol efflux to serum were significantly impaired in all three groups of diabetic patients (p < 0.01). SAA inversely correlated with SR-BI-mediated cholesterol efflux (r ¼ 0.36, p < 0.01) but did not correlate with ABCG1-mediated cholesterol efflux. Stepwise linear regression analysis showed that HDL, the presence or absence of diabetes, and log(SAA) were significant independent determinants of SR-BI-mediated cholesterol efflux to serum. Conclusion: SAA was increased in type 2 diabetic patients with incipient or overt nephropathy, and SAA was associated with impairment of SR-BI-mediated cholesterol efflux to serum. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Serum amyloid A Cellular cholesterol efflux Scavenger receptor class B type I ATP-binding cassette transporter G1 High density lipoprotein Reverse cholesterol transport
1. Introduction Serum amyloid A (SAA) is an acute phase response protein and is a sensitive marker of the acute inflammatory response. It has been suggested that SAA may be involved in the defence mechanisms against pathogens and may function as an effector molecule of the immune system [1]. SAA has apolipoprotein properties and is mainly associated with high density lipoprotein (HDL) in the circulation. Both SAA and apolipoprotein AI (apo AI) share the same amphipathic helical structure, and because of its association with HDL, SAA may also play a role in lipoprotein metabolism [2,3]. Level of SAA increases up to 1000-fold during acute phase inflammation and SAA can displace apo AI from the phospholipid surface of HDL, thus altering the structures and functions of HDL [3,4]. There are conflicting data on the impact of enrichment of HDL with SAA on the property of HDL to induce cholesterol efflux, with some studies reporting a reduction in the ability of HDL to efflux cholesterol from * Corresponding author. Tel.: þ852 2255 5859; fax: þ852 2816 2187. E-mail address: [email protected] (K.C.B. Tan). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.10.008
cells in vitro [5,6] whilst others have shown the contrary [7e9]. The effect of SAA on cholesterol efflux to HDL seems to be partly dependent on the experimental conditions used and the cholesterol transporters involved. Using a mouse model, Annema et al. has recently reported that SAA impaired reverse cholesterol transport during the acute phase response in vivo [10]. The impact of SAA on reverse cholesterol transport has been mainly investigated in acute inflammatory state but the effect of SAA is much less clear in chronic inflammation where the concentration of SAA is much lower. Type 2 diabetes mellitus is associated with chronic subclinical low grade inflammation [11]. We have previously reported that cellular cholesterol efflux to serum in patients with type 2 diabetes is impaired [12]. The capacity of serum to induce cellular cholesterol efflux is influenced not only by the concentrations of lipoproteins, like HDL, that act as cholesterol acceptors but also by other serum components. Hence, we have determined firstly whether there are any significant changes in SAA levels in type 2 diabetic patients with and without nephropathy and secondly, the relationship between SAA and serum capacity to induce cellular cholesterol efflux.
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2. Methods
2.4. Biochemical assays
2.1. Subjects
Serum amyloid A was measured by commercial sandwich ELISA kit (abcam, San Francisco, CA, USA). Plasma total cholesterol and triglyceride were determined enzymatically on a Hitachi 912 analyser (Roche Diagnostics GmbH, Mannheim, Germany). HDLcholesterol was measured using a homogenous method with polyethylene glycol-modified enzymes and alpha-cyclodextrin. Plasma apo AI and apo B were measured by rate nephelometry using the Beckman Array System (Beckman Coulter, Brea, CA, USA). HbA1c was measured in whole blood samples using ion-exchange high performance liquid chromatography with the Bio-Rad Variant Haemoglobin Testing System (Bio-Rad, Hercules, CA, USA). Plasma creatinine was measured by the Jaffe method. Urinary albumin excretion rate was determined from the mean of two consecutive 12-h overnight urine collections. Urine albumin was measured by rate nephelometry using the Beckman Array 360 Analyser (Beckman Coulter, Brea, CA, USA). Estimated glomerular filtration rate (eGFR) was calculated by Modification of Diet in Renal Disease (MDRD) Study equation.
Type 2 diabetic patients were recruited from the diabetes clinics at Queen Mary Hospital in Hong Kong. Diabetic patients were invited to participate when they attended their annual screening visit for diabetic complications and were subdivided into those with normoalbuminuria (300 mg/day) according to their urinary albumin excretion rate. Patients with history of cardiovascular disease and/or receiving lipid lowering agents were excluded. Recent acute illness was also an exclusion criterion. Healthy nondiabetic controls were recruited from the community. Blood samples were collected after an overnight fast for the measurements of HbA1c, glucose, renal function, lipids, SAA, and cellular cholesterol efflux to serum. The study was approved by the Ethics Committee of the University of Hong Kong, and informed consent was obtained from all subjects. 2.2. Ex vivo cellular cholesterol efflux to serum
2.5. HDL binding assay Scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporters G1 (ABCG1) are the main cholesterol transporters that mediate cholesterol efflux to HDL. To measure the SRBI-mediated cellular cholesterol efflux to serum and ABCG1mediated cellular cholesterol efflux to serum, Fu5AH rat hepatoma cells which express only SR-BI but no functional ABCs [13], and human ABCG1-transfected Chinese Hamster Ovary-K1 (CHOK1) cells (a generous gift from Prof. W. Jessup) were used respectively [14]. Cells were cultured in minimum essential medium (MEM) and Ham’s F-12K medium respectively (Gibco, Grand Island, NY, USA), which were also supplemented with 5% FBS. Eighty per cent confluent cells were labelled with [3H]-cholesterol for 18 h (37kBq/well; Amersham Biosciences, Pittsburgh, PA, USA). After that, the medium was removed and cells were incubated with fresh medium containing 0.5% BSA for a further 4 h to allow the incorporated [3H]-cholesterol to equilibrate among the cellular cholesterol pools. Cells were then washed with PBS and incubated with serum samples from either diabetic patients or healthy controls (diluted 1:20 with medium) for 6 h at 37 C. Supernatants were collected and cells were lysed with 0.1% Triton X-100 in PBS (500 ml). The amount of [3H]-cholesterol in each fraction was assessed by scintillation counting using a Packard scintillation counter (Perkin Elmer, Waltham, MA, USA). Cholesterol efflux was calculated as the percentage of [3H]-cholesterol recovered in the supernatant compared to the total cellular [3H]-label for each sample. All samples were assayed in duplicate, and inter-assay coefficients of variation for SR-BI-mediated cholesterol efflux assay and ABCG1-mediated cholesterol efflux assay was 8.0% and 9.7% respectively.
Fu5AH rat hepatoma cells were seeded in 24-well plate until confluent in MEM with 5% FBS. Cells were then fasted with plain medium for 16 h and incubated with ice-cold MEM with various concentrations of lipid-free SAA (Peprotech, Rocky Hill, NJ) for 2 h in 4 C. The medium was removed and 5 mg/ml DiI-HDL (BTI, Stoughton, MA) in MEM was added and incubated for 2 h in 4 C. Cells were then washed once with ice-cold PBS and lysed with 0.1% Triton X-100 in PBS (200 ml). The fluorescence intensity of the cell lysate reflecting the amount of bound HDL was measured on black 96-well microtiter plate (Costar, Palo Alto, CA), with excitation and emission wavelengths set at 520 and 578 nm respectively by the Tecan plate reader (Männedorf, Schweiz). Results were expressed as percentage of the control value obtained in the absence of SAA. Each of the binding experiment shown is representative of the duplicate results obtained in three individual experiments. 2.6. Statistics Numerical data were expressed as mean standard deviation of the mean (SD) or median (inter-quartile range). Data that were not normally distributed were logarithmically transformed before analyses were made. Analysis of variance (ANOVA) was used to compare continuous variables for multiple groups followed by posthoc multiple comparisons using Dunnett t-tests with the nondiabetic control as the reference group. Pearson’s correlations were used to test the relationship between variables, and multiple stepwise linear regression analysis was used to assess the relationships between cholesterol efflux to serum and various variables simultaneously.
2.3. Ex vivo cellular cholesterol efflux to HDL 3. Results Protocol for cholesterol efflux to HDL was similar to that of serum, but HDL (25 mg/ml HDL-protein) was used as the cholesterol acceptor for each sample. To prepare HDL from plasma, a one-step ultracentrifugation protocol was used. Briefly, plasma was first salted with KBr to establish a density at 1.24 g/ml and was overlaid on top with a single gradient of 1.076 g/ml NaBr solution. Mixed plasma was then ultracentrifuged (Beckman Coulter, Brea, CA, USA) for 3.5 h at 100,000 rpm at 10 C and purified HDL (1.063e1.21 g/ml) was extracted from the tube. Lastly, HDL was desalted with Econo-PacÒ 10DG desalting column (Bio-Rad, Hercules, CA, USA). Protein concentration was measured by the method of Lowry et al. [15].
The clinical characteristics of control and type 2 diabetic patients are shown in Table 1. Control subjects were younger than the diabetic patients. All three groups of diabetic patients had larger waist circumference and higher systolic blood pressure. Glycaemic control was comparable in the three groups of diabetic patients (Table 2) and none of the patients was on pioglitazone which could affect HDL efflux capacity. Diabetic patients had hypertriglyceridaemia and low plasma HDL-cholesterol (HDL-C) and apo AI compared to healthy controls. Serum amyloid A was significantly higher in diabetic patients with microalbuminuria or proteinuria
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Table 1 Clinical characteristics of subjects.
Age (years) Male/female (%) Duration of diabetes (years) Hypertension (%) Retinopathy (%) Smoker (%) Insulin therapy (%) BMI (kg/m2) Waist (cm) BP systolic (mmHg) BP diastolic (mmHg)
Control (n ¼ 275)
Normoalbuminuria (n ¼ 110)
Microalbuminuria (n ¼ 79)
Proteinuria (n ¼ 75)
46.2 8.5 47/53 e e e 13 e 24.4 3.4 81.2 9.7 117.0 17.3 75.5 10.3
50.4 8.4** 43/67 12.4 6.6 45 25 9.1 24 25.2 3.7 84.1 11.5* 122.1 20.6* 75.4 8.5
56.6 10.2** 63/37 11.5 8.2 77 51 7.6 49 26.9 4.3** 90.6 11.1** 135.5 20.8** 78.1 8.4
57.0 10.3** 80/20 11.7 5.8 84 71 12 58 26.0 3.8** 91.5 10.4** 140.2 27.6** 79.1 10.6*
Data are expressed as mean SD. *p < 0.05 and **p < 0.01 versus healthy controls.
than controls whereas in the normoalbuminuric patients, SAA was also increased but did not reach statistical significance on post-hoc analysis. Cholesterol efflux to serum mediated by SR-BI, and ABCG1 was impaired in diabetic patients (Fig. 1a and b respectively). Since plasma HDL-C levels differed significantly between diabetic patients and controls, data were also analysed after adjusting for HDLC level and other potential confounders including age, gender, BMI and smoking. The differences in cholesterol efflux remained significant. As expected, both SR-BI and ABCG1-mediated cholesterol efflux to serum correlated with plasma HDL-C (r ¼ 0.44, p < 0.01 and r ¼ 0.19, p < 0.01 respectively) and with apo AI (r ¼ 0.49, p < 0.01 and r ¼ 0.22, p < 0.01 respectively), and the association was much stronger with SR-BI-mediated cholesterol efflux. Both SR-BI and ABCG1-mediated cholesterol efflux to serum also correlated with log(eGFR) (r ¼ 0.23, p < 0.01 and r ¼ 0.16, p < 0.01 respectively) but no association was seen with HbA1c and other plasma lipid parameters. There was an inverse correlation between SR-BImediated cholesterol efflux to serum and log(SAA) (r ¼ 0.36, p < 0.01) (Fig. 2a) but no correlation was seen with ABCG1mediated cholesterol efflux (Fig. 2b). The association between SRBI-mediated cholesterol efflux to serum and log(SAA) was still significant when the diabetic cohort was analysed alone (r ¼ 0.28, p < 0.01) and was similar in all three diabetic subgroups. Forward stepwise linear regression analysis including age, sex, BMI, smoking, the presence or absence of diabetes, HDL-C, log(SAA) and log(eGFR) showed that only HDL-C, the presence or absence of diabetes, and log(SAA) were significant independent determinants of SR-BI-mediated cholesterol efflux to serum, accounting for 24%, 11% and 9% of variation in SR-BI-mediated cholesterol efflux to serum respectively. For ABCG1-mediated cholesterol efflux to serum, HDL-C was the only significant independent determinant. Since SAA was significantly associated with SR-BI-mediated cholesterol efflux to serum, we proceeded to determine whether this was related to the effect of SAA on HDL function. High SAA concentration during acute phase response has been shown to affect HDL efflux capacity [5], but whether the much lower level of SAA seen in our diabetic subjects without acute illness has any effect on HDL is unclear. The amount of SAA in HDL (HDL-SAA) was measured in 22 randomly selected diabetic subjects from different quartiles of SAA in serum and the result was shown in Fig. 3a. There was a significant correlation between HDL-SAA and SAA in serum (r ¼ 0.49, p < 0.05). The ability of the corresponding HDL to induce cholesterol efflux was similar when diabetic subjects with HDL-SAA in the top quartile were compared with those in the bottom quartile (14.6 3.1% versus 14.3 2.9% respectively). In addition, there was no correlation between HDL-SAA and the ability of the corresponding HDL in diabetic patients to induce cholesterol efflux from Fu5AH cells. Since SAA could act as a ligand of SR-BI [16], binding assay was performed to investigate whether lipid-free SAA affected HDL
binding to SR-BI. Binding of HDL to SR-BI was inhibited in a dosedependent manner within the physiological range of SAA observed in our study (Fig. 3b), resulting in reduced efflux. Taken together, this would suggest that the association between SAA and SR-BI-mediated cholesterol efflux to serum was unlikely related to the degree of SAA enrichment of HDL, and might partly be due to the effect of lipid-free SAA. 4. Discussion Efflux of free cholesterol from cells is an early step of reverse cholesterol transport and can occur by a number of mechanisms which include regulated transporter-facilitated processes as well as aqueous diffusion. The main cholesterol transporters involved in cellular cholesterol efflux include ABCA1, ABCG1 and SR-BI [17,18]. All three transporters are expressed in macrophages. ABCA1 mediates cholesterol efflux to lipid-free apo AI and pre-b HDL whereas both ABCG1 and SR-BI mediate cholesterol efflux to mature HDL. Khera et al. have demonstrated that cholesterol efflux capacity is associated with atherosclerosis in humans [19], and Pajunen P. et al. have shown that cholesterol efflux capacity to plasma predicted the severity and extent of coronary artery disease in type 2 diabetic patients [20]. We have previously reported that serum capacity to induce ABCA1-mediated cholesterol efflux as well as SR-BImediated cholesterol efflux were decreased in patients with type 2 diabetes mellitus [12]. We have further shown in the present study that serum capacity to induce ABCG1-mediated cholesterol efflux was also impaired in diabetic patients. Although the differences in ABCG1 and SR-BI-mediated cholesterol efflux between type 2 diabetic patients and controls might seem small, the magnitude is similar to the previously reported differences in cholesterol efflux capacity between patients with coronary heart disease and matched controls [19,21]. The greatest abnormalities were observed in diabetic subjects with overt nephropathy and serum cholesterol efflux capacity correlated with eGFR. This might be partly related to the lower plasma levels of HDL in patients with nephropathy as well as the qualitative changes in HDL seen in uremic condition causing HDL dysfunction [22]. Apart from the changes in HDL, other serum components can also influence the ability of serum to induce cholesterol efflux from cells. We have evaluated the role of SAA in patients with type 2 diabetes mellitus. Even in the absence of any acute illness, SAA was significantly elevated in type 2 diabetic patients with incipient or overt nephropathy. This is similar to the results of Dalla Vestra et al. who reported that SAA correlated with albumin excretion rate in patients with type 2 diabetes [23]. Serum amyloid A is part of a systemic response to injury to recycle and reuse cholesterol from damaged cells. This physiological response may become maladaptive and chronic modest elevation in SAA may have different biological effects than the massive elevations seen in acute
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Table 2 Glycaemic control, renal function, plasma lipids, and SAA.
HbA1c (%/mmol/mol) Fasting glucose (mmol/L) eGFR (mL/min/1.73 m2) Total Cholesterol (mmol/L) Triglyceride (mmol/L) LDL-Cholesterol (mmol/L) HDL-Cholesterol (mmol/L) Apo AI (g/L) Apo B (g/L) SAA (ng/ml)
Control (n ¼ 275)
Normoalbuminuria (n ¼ 110)
Microalbuminuria (n ¼ 79)
Proteinuria (n ¼ 75)
5.6 0.5 5.0 0.7 78 (69e89) 4.9 0.9 1.0 (0.7e1.3) 3.0 0.8 1.4 0.4 1.41 0.28 0.89 0.21 106.5 (79.4e137.2)
8.2 1.3** 8.4 2.0** 74 (64e82)* 4.8 0.8 1.2 (0.9e1.8) 2.9 0.7 1.3 0.3* 1.33 0.22 0.87 0.19 115.6 (66.1e151.1)
8.5 1.7** 9.2 4.3** 59 (45e70)** 4.8 0.9 1.6 (1.2e2.2)** 2.8 0.8 1.2 0.4** 1.28 0.22* 0.91 0.19 126.9 (91.7e218.7)**
8.5 1.7** 8.0 3.0** 51 (36e68)** 5.0 0.9 1.5 (1.0e2.4)** 3.0 0.8 1.2 0.4** 1.27 0.25** 0.98 0.24* 127.5 (83.8e170.4)**
Data are expressed as mean SD or median (interquartile range). *p < 0.05 and **p < 0.01 versus healthy controls.
inflammatory response. For example, there is epidemiological data linking SAA with cardiovascular disease [24,25], and it has also been shown that SAA may be proatherogenic [26]. In our study, SAA is a significant determinant of SR-BI-mediated cholesterol efflux to serum, and our experimental data suggest that this is unlikely to be related to HDL enrichment with SAA. In acute phase HDL, SAA may account for up to 17%e87% of the total apolipoproteins present in HDL [27], and Banka et al. reported that the cholesterol efflux property of HDL was only reduced when SAA constituted more than 50% of HDL protein [5]. The level of SAA in serum was much lower in our diabetic subjects than that seen in acute phase response. Even in subjects in the highest quartile of SAA in serum, HDL-SAA accounted for less than 5% of HDL protein. Hence, the degree of SAA enrichment of HDL was not sufficient to alter the ability of HDL to act as cholesterol acceptor and unlikely to
Fig. 1. SR-BI-mediated cholesterol efflux to serum (1a) and ABCG1-mediated cholesterol efflux to serum (1b) in controls and diabetic subjects. Data are expressed as mean SD. *p < 0.05 compared with healthy controls.
account for the impairment in SR-BI-mediated cholesterol efflux to serum. Lipid-free SAA is not a cholesterol acceptor for SR-BI [28], but can act as a ligand to SR-BI [16]. Results from our binding assay suggest that lipid-free SAA can inhibit the binding of HDL to SR-BI and is similar to the findings of Cai et al. [16]. Since SR-BI-mediated cholesterol efflux from cells to HDL requires the binding of HDL to SR-BI particularly at low acceptor concentration [29], inhibition of
Fig. 2. Correlation between SAA and SR-BI-mediated cholesterol efflux to serum, r ¼ 0.36, p < 0.01 (2a), and ABCG1-mediated cholesterol efflux to serum (2b).
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In conclusion, SAA was increased in type 2 diabetic patients with incipient or overt nephropathy, and SAA was associated with impairment of SR-BI-mediated cholesterol efflux to serum. Financial support This study is supported by funding from the Hong Kong Research Grants Council Research Fund [HKU 776709M]. Conflict of interest The authors have no conflict of interest to disclose. References
Fig. 3. Correlation between SAA and HDL-SAA in diabetic subjects, r ¼ 0.49, p < 0.05 (3a). Effect of lipid-free human SAA on HDL binding to SR-BI in Fu5AH cells (3b).
HDL binding by lipid-free SAA may lead to impairment of cholesterol efflux. In addition, SAA has an inhibitory effect on LCAT activity [30], which may also lead to impairment in cholesterol efflux to serum [18]. We did not find any association between SAA and ABCG1-mediated cholesterol efflux and this is in keeping with experimental data showing that ABCG1-dependent cholesterol efflux was independent of SAA during inflammation [31]. Our study has several limitations. The control subjects were significantly younger than the diabetic patients, and we have adjusted for age as a confounding factor in our analysis. Because of its cross-sectional design, we can only demonstrate associations and not causal relationships. We have only investigated the effect of SAA on cholesterol efflux capacity of serum which reflects the ability to mobilise free cholesterol from cells. Although it has been shown that cholesterol efflux capacity is associated with atherosclerosis in humans [19], we have not evaluated other components of the reverse cholesterol pathway. Another major limitation is that we have not included ABCA1-mediated cholesterol efflux to serum in the present study, and we cannot comment on the overall effect of SAA on cholesterol efflux derangement in type 2 diabetic patients. Work is on-going and our preliminary data on ABCA1mediated cholesterol efflux to serum have so far shown that ABCA1-mediated cholesterol efflux to serum was significantly impaired in diabetic patients with proteinuria (unpublished data) which was in keeping with our earlier study [12].
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Impact of serum amyloid A on cellular cholesterol efflux to serum in type 2 diabetes mellitus J.G.S. Tsun, S.W.M. Shiu, Y. Wong, S. Yung, T.M. Chan, K.C.B. Tan* Department of Medicine, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 May 2013 Received in revised form 7 September 2013 Accepted 10 October 2013 Available online 18 October 2013
Objective: Serum amyloid A (SAA) is an acute phase response protein and has apolipoprotein properties. Since type 2 diabetes is associated with chronic subclinical inflammation, the objective of this study is to investigate the changes in SAA level in type 2 diabetic patients and to evaluate the relationship between SAA and the capacity of serum to induce cellular cholesterol efflux via the two known cholesterol transporters, scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter G1 (ABCG1). Methods: 264 patients with type 2 diabetes mellitus (42% with normoalbuminuria, 30% microalbuminuria, and 28% proteinuria) and 275 non-diabetic controls were recruited. SAA was measured by ELISA. SR-BI and ABCG1-mediated cholesterol efflux to serum were determined by measuring the transfer of [3H]cholesterol from Fu5AH rat hepatoma cells expressing SR-BI and from human ABCG1transfected CHO-K1 cells to the medium containing the tested serum respectively. Results: SAA was significantly increased in diabetic patients with incipient or overt nephropathy. Both SR-BI and ABCG1-mediated cholesterol efflux to serum were significantly impaired in all three groups of diabetic patients (p < 0.01). SAA inversely correlated with SR-BI-mediated cholesterol efflux (r ¼ 0.36, p < 0.01) but did not correlate with ABCG1-mediated cholesterol efflux. Stepwise linear regression analysis showed that HDL, the presence or absence of diabetes, and log(SAA) were significant independent determinants of SR-BI-mediated cholesterol efflux to serum. Conclusion: SAA was increased in type 2 diabetic patients with incipient or overt nephropathy, and SAA was associated with impairment of SR-BI-mediated cholesterol efflux to serum. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Serum amyloid A Cellular cholesterol efflux Scavenger receptor class B type I ATP-binding cassette transporter G1 High density lipoprotein Reverse cholesterol transport
1. Introduction Serum amyloid A (SAA) is an acute phase response protein and is a sensitive marker of the acute inflammatory response. It has been suggested that SAA may be involved in the defence mechanisms against pathogens and may function as an effector molecule of the immune system [1]. SAA has apolipoprotein properties and is mainly associated with high density lipoprotein (HDL) in the circulation. Both SAA and apolipoprotein AI (apo AI) share the same amphipathic helical structure, and because of its association with HDL, SAA may also play a role in lipoprotein metabolism [2,3]. Level of SAA increases up to 1000-fold during acute phase inflammation and SAA can displace apo AI from the phospholipid surface of HDL, thus altering the structures and functions of HDL [3,4]. There are conflicting data on the impact of enrichment of HDL with SAA on the property of HDL to induce cholesterol efflux, with some studies reporting a reduction in the ability of HDL to efflux cholesterol from * Corresponding author. Tel.: þ852 2255 5859; fax: þ852 2816 2187. E-mail address: [email protected] (K.C.B. Tan). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.10.008
cells in vitro [5,6] whilst others have shown the contrary [7e9]. The effect of SAA on cholesterol efflux to HDL seems to be partly dependent on the experimental conditions used and the cholesterol transporters involved. Using a mouse model, Annema et al. has recently reported that SAA impaired reverse cholesterol transport during the acute phase response in vivo [10]. The impact of SAA on reverse cholesterol transport has been mainly investigated in acute inflammatory state but the effect of SAA is much less clear in chronic inflammation where the concentration of SAA is much lower. Type 2 diabetes mellitus is associated with chronic subclinical low grade inflammation [11]. We have previously reported that cellular cholesterol efflux to serum in patients with type 2 diabetes is impaired [12]. The capacity of serum to induce cellular cholesterol efflux is influenced not only by the concentrations of lipoproteins, like HDL, that act as cholesterol acceptors but also by other serum components. Hence, we have determined firstly whether there are any significant changes in SAA levels in type 2 diabetic patients with and without nephropathy and secondly, the relationship between SAA and serum capacity to induce cellular cholesterol efflux.
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2. Methods
2.4. Biochemical assays
2.1. Subjects
Serum amyloid A was measured by commercial sandwich ELISA kit (abcam, San Francisco, CA, USA). Plasma total cholesterol and triglyceride were determined enzymatically on a Hitachi 912 analyser (Roche Diagnostics GmbH, Mannheim, Germany). HDLcholesterol was measured using a homogenous method with polyethylene glycol-modified enzymes and alpha-cyclodextrin. Plasma apo AI and apo B were measured by rate nephelometry using the Beckman Array System (Beckman Coulter, Brea, CA, USA). HbA1c was measured in whole blood samples using ion-exchange high performance liquid chromatography with the Bio-Rad Variant Haemoglobin Testing System (Bio-Rad, Hercules, CA, USA). Plasma creatinine was measured by the Jaffe method. Urinary albumin excretion rate was determined from the mean of two consecutive 12-h overnight urine collections. Urine albumin was measured by rate nephelometry using the Beckman Array 360 Analyser (Beckman Coulter, Brea, CA, USA). Estimated glomerular filtration rate (eGFR) was calculated by Modification of Diet in Renal Disease (MDRD) Study equation.
Type 2 diabetic patients were recruited from the diabetes clinics at Queen Mary Hospital in Hong Kong. Diabetic patients were invited to participate when they attended their annual screening visit for diabetic complications and were subdivided into those with normoalbuminuria (300 mg/day) according to their urinary albumin excretion rate. Patients with history of cardiovascular disease and/or receiving lipid lowering agents were excluded. Recent acute illness was also an exclusion criterion. Healthy nondiabetic controls were recruited from the community. Blood samples were collected after an overnight fast for the measurements of HbA1c, glucose, renal function, lipids, SAA, and cellular cholesterol efflux to serum. The study was approved by the Ethics Committee of the University of Hong Kong, and informed consent was obtained from all subjects. 2.2. Ex vivo cellular cholesterol efflux to serum
2.5. HDL binding assay Scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporters G1 (ABCG1) are the main cholesterol transporters that mediate cholesterol efflux to HDL. To measure the SRBI-mediated cellular cholesterol efflux to serum and ABCG1mediated cellular cholesterol efflux to serum, Fu5AH rat hepatoma cells which express only SR-BI but no functional ABCs [13], and human ABCG1-transfected Chinese Hamster Ovary-K1 (CHOK1) cells (a generous gift from Prof. W. Jessup) were used respectively [14]. Cells were cultured in minimum essential medium (MEM) and Ham’s F-12K medium respectively (Gibco, Grand Island, NY, USA), which were also supplemented with 5% FBS. Eighty per cent confluent cells were labelled with [3H]-cholesterol for 18 h (37kBq/well; Amersham Biosciences, Pittsburgh, PA, USA). After that, the medium was removed and cells were incubated with fresh medium containing 0.5% BSA for a further 4 h to allow the incorporated [3H]-cholesterol to equilibrate among the cellular cholesterol pools. Cells were then washed with PBS and incubated with serum samples from either diabetic patients or healthy controls (diluted 1:20 with medium) for 6 h at 37 C. Supernatants were collected and cells were lysed with 0.1% Triton X-100 in PBS (500 ml). The amount of [3H]-cholesterol in each fraction was assessed by scintillation counting using a Packard scintillation counter (Perkin Elmer, Waltham, MA, USA). Cholesterol efflux was calculated as the percentage of [3H]-cholesterol recovered in the supernatant compared to the total cellular [3H]-label for each sample. All samples were assayed in duplicate, and inter-assay coefficients of variation for SR-BI-mediated cholesterol efflux assay and ABCG1-mediated cholesterol efflux assay was 8.0% and 9.7% respectively.
Fu5AH rat hepatoma cells were seeded in 24-well plate until confluent in MEM with 5% FBS. Cells were then fasted with plain medium for 16 h and incubated with ice-cold MEM with various concentrations of lipid-free SAA (Peprotech, Rocky Hill, NJ) for 2 h in 4 C. The medium was removed and 5 mg/ml DiI-HDL (BTI, Stoughton, MA) in MEM was added and incubated for 2 h in 4 C. Cells were then washed once with ice-cold PBS and lysed with 0.1% Triton X-100 in PBS (200 ml). The fluorescence intensity of the cell lysate reflecting the amount of bound HDL was measured on black 96-well microtiter plate (Costar, Palo Alto, CA), with excitation and emission wavelengths set at 520 and 578 nm respectively by the Tecan plate reader (Männedorf, Schweiz). Results were expressed as percentage of the control value obtained in the absence of SAA. Each of the binding experiment shown is representative of the duplicate results obtained in three individual experiments. 2.6. Statistics Numerical data were expressed as mean standard deviation of the mean (SD) or median (inter-quartile range). Data that were not normally distributed were logarithmically transformed before analyses were made. Analysis of variance (ANOVA) was used to compare continuous variables for multiple groups followed by posthoc multiple comparisons using Dunnett t-tests with the nondiabetic control as the reference group. Pearson’s correlations were used to test the relationship between variables, and multiple stepwise linear regression analysis was used to assess the relationships between cholesterol efflux to serum and various variables simultaneously.
2.3. Ex vivo cellular cholesterol efflux to HDL 3. Results Protocol for cholesterol efflux to HDL was similar to that of serum, but HDL (25 mg/ml HDL-protein) was used as the cholesterol acceptor for each sample. To prepare HDL from plasma, a one-step ultracentrifugation protocol was used. Briefly, plasma was first salted with KBr to establish a density at 1.24 g/ml and was overlaid on top with a single gradient of 1.076 g/ml NaBr solution. Mixed plasma was then ultracentrifuged (Beckman Coulter, Brea, CA, USA) for 3.5 h at 100,000 rpm at 10 C and purified HDL (1.063e1.21 g/ml) was extracted from the tube. Lastly, HDL was desalted with Econo-PacÒ 10DG desalting column (Bio-Rad, Hercules, CA, USA). Protein concentration was measured by the method of Lowry et al. [15].
The clinical characteristics of control and type 2 diabetic patients are shown in Table 1. Control subjects were younger than the diabetic patients. All three groups of diabetic patients had larger waist circumference and higher systolic blood pressure. Glycaemic control was comparable in the three groups of diabetic patients (Table 2) and none of the patients was on pioglitazone which could affect HDL efflux capacity. Diabetic patients had hypertriglyceridaemia and low plasma HDL-cholesterol (HDL-C) and apo AI compared to healthy controls. Serum amyloid A was significantly higher in diabetic patients with microalbuminuria or proteinuria
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Table 1 Clinical characteristics of subjects.
Age (years) Male/female (%) Duration of diabetes (years) Hypertension (%) Retinopathy (%) Smoker (%) Insulin therapy (%) BMI (kg/m2) Waist (cm) BP systolic (mmHg) BP diastolic (mmHg)
Control (n ¼ 275)
Normoalbuminuria (n ¼ 110)
Microalbuminuria (n ¼ 79)
Proteinuria (n ¼ 75)
46.2 8.5 47/53 e e e 13 e 24.4 3.4 81.2 9.7 117.0 17.3 75.5 10.3
50.4 8.4** 43/67 12.4 6.6 45 25 9.1 24 25.2 3.7 84.1 11.5* 122.1 20.6* 75.4 8.5
56.6 10.2** 63/37 11.5 8.2 77 51 7.6 49 26.9 4.3** 90.6 11.1** 135.5 20.8** 78.1 8.4
57.0 10.3** 80/20 11.7 5.8 84 71 12 58 26.0 3.8** 91.5 10.4** 140.2 27.6** 79.1 10.6*
Data are expressed as mean SD. *p < 0.05 and **p < 0.01 versus healthy controls.
than controls whereas in the normoalbuminuric patients, SAA was also increased but did not reach statistical significance on post-hoc analysis. Cholesterol efflux to serum mediated by SR-BI, and ABCG1 was impaired in diabetic patients (Fig. 1a and b respectively). Since plasma HDL-C levels differed significantly between diabetic patients and controls, data were also analysed after adjusting for HDLC level and other potential confounders including age, gender, BMI and smoking. The differences in cholesterol efflux remained significant. As expected, both SR-BI and ABCG1-mediated cholesterol efflux to serum correlated with plasma HDL-C (r ¼ 0.44, p < 0.01 and r ¼ 0.19, p < 0.01 respectively) and with apo AI (r ¼ 0.49, p < 0.01 and r ¼ 0.22, p < 0.01 respectively), and the association was much stronger with SR-BI-mediated cholesterol efflux. Both SR-BI and ABCG1-mediated cholesterol efflux to serum also correlated with log(eGFR) (r ¼ 0.23, p < 0.01 and r ¼ 0.16, p < 0.01 respectively) but no association was seen with HbA1c and other plasma lipid parameters. There was an inverse correlation between SR-BImediated cholesterol efflux to serum and log(SAA) (r ¼ 0.36, p < 0.01) (Fig. 2a) but no correlation was seen with ABCG1mediated cholesterol efflux (Fig. 2b). The association between SRBI-mediated cholesterol efflux to serum and log(SAA) was still significant when the diabetic cohort was analysed alone (r ¼ 0.28, p < 0.01) and was similar in all three diabetic subgroups. Forward stepwise linear regression analysis including age, sex, BMI, smoking, the presence or absence of diabetes, HDL-C, log(SAA) and log(eGFR) showed that only HDL-C, the presence or absence of diabetes, and log(SAA) were significant independent determinants of SR-BI-mediated cholesterol efflux to serum, accounting for 24%, 11% and 9% of variation in SR-BI-mediated cholesterol efflux to serum respectively. For ABCG1-mediated cholesterol efflux to serum, HDL-C was the only significant independent determinant. Since SAA was significantly associated with SR-BI-mediated cholesterol efflux to serum, we proceeded to determine whether this was related to the effect of SAA on HDL function. High SAA concentration during acute phase response has been shown to affect HDL efflux capacity [5], but whether the much lower level of SAA seen in our diabetic subjects without acute illness has any effect on HDL is unclear. The amount of SAA in HDL (HDL-SAA) was measured in 22 randomly selected diabetic subjects from different quartiles of SAA in serum and the result was shown in Fig. 3a. There was a significant correlation between HDL-SAA and SAA in serum (r ¼ 0.49, p < 0.05). The ability of the corresponding HDL to induce cholesterol efflux was similar when diabetic subjects with HDL-SAA in the top quartile were compared with those in the bottom quartile (14.6 3.1% versus 14.3 2.9% respectively). In addition, there was no correlation between HDL-SAA and the ability of the corresponding HDL in diabetic patients to induce cholesterol efflux from Fu5AH cells. Since SAA could act as a ligand of SR-BI [16], binding assay was performed to investigate whether lipid-free SAA affected HDL
binding to SR-BI. Binding of HDL to SR-BI was inhibited in a dosedependent manner within the physiological range of SAA observed in our study (Fig. 3b), resulting in reduced efflux. Taken together, this would suggest that the association between SAA and SR-BI-mediated cholesterol efflux to serum was unlikely related to the degree of SAA enrichment of HDL, and might partly be due to the effect of lipid-free SAA. 4. Discussion Efflux of free cholesterol from cells is an early step of reverse cholesterol transport and can occur by a number of mechanisms which include regulated transporter-facilitated processes as well as aqueous diffusion. The main cholesterol transporters involved in cellular cholesterol efflux include ABCA1, ABCG1 and SR-BI [17,18]. All three transporters are expressed in macrophages. ABCA1 mediates cholesterol efflux to lipid-free apo AI and pre-b HDL whereas both ABCG1 and SR-BI mediate cholesterol efflux to mature HDL. Khera et al. have demonstrated that cholesterol efflux capacity is associated with atherosclerosis in humans [19], and Pajunen P. et al. have shown that cholesterol efflux capacity to plasma predicted the severity and extent of coronary artery disease in type 2 diabetic patients [20]. We have previously reported that serum capacity to induce ABCA1-mediated cholesterol efflux as well as SR-BImediated cholesterol efflux were decreased in patients with type 2 diabetes mellitus [12]. We have further shown in the present study that serum capacity to induce ABCG1-mediated cholesterol efflux was also impaired in diabetic patients. Although the differences in ABCG1 and SR-BI-mediated cholesterol efflux between type 2 diabetic patients and controls might seem small, the magnitude is similar to the previously reported differences in cholesterol efflux capacity between patients with coronary heart disease and matched controls [19,21]. The greatest abnormalities were observed in diabetic subjects with overt nephropathy and serum cholesterol efflux capacity correlated with eGFR. This might be partly related to the lower plasma levels of HDL in patients with nephropathy as well as the qualitative changes in HDL seen in uremic condition causing HDL dysfunction [22]. Apart from the changes in HDL, other serum components can also influence the ability of serum to induce cholesterol efflux from cells. We have evaluated the role of SAA in patients with type 2 diabetes mellitus. Even in the absence of any acute illness, SAA was significantly elevated in type 2 diabetic patients with incipient or overt nephropathy. This is similar to the results of Dalla Vestra et al. who reported that SAA correlated with albumin excretion rate in patients with type 2 diabetes [23]. Serum amyloid A is part of a systemic response to injury to recycle and reuse cholesterol from damaged cells. This physiological response may become maladaptive and chronic modest elevation in SAA may have different biological effects than the massive elevations seen in acute
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Table 2 Glycaemic control, renal function, plasma lipids, and SAA.
HbA1c (%/mmol/mol) Fasting glucose (mmol/L) eGFR (mL/min/1.73 m2) Total Cholesterol (mmol/L) Triglyceride (mmol/L) LDL-Cholesterol (mmol/L) HDL-Cholesterol (mmol/L) Apo AI (g/L) Apo B (g/L) SAA (ng/ml)
Control (n ¼ 275)
Normoalbuminuria (n ¼ 110)
Microalbuminuria (n ¼ 79)
Proteinuria (n ¼ 75)
5.6 0.5 5.0 0.7 78 (69e89) 4.9 0.9 1.0 (0.7e1.3) 3.0 0.8 1.4 0.4 1.41 0.28 0.89 0.21 106.5 (79.4e137.2)
8.2 1.3** 8.4 2.0** 74 (64e82)* 4.8 0.8 1.2 (0.9e1.8) 2.9 0.7 1.3 0.3* 1.33 0.22 0.87 0.19 115.6 (66.1e151.1)
8.5 1.7** 9.2 4.3** 59 (45e70)** 4.8 0.9 1.6 (1.2e2.2)** 2.8 0.8 1.2 0.4** 1.28 0.22* 0.91 0.19 126.9 (91.7e218.7)**
8.5 1.7** 8.0 3.0** 51 (36e68)** 5.0 0.9 1.5 (1.0e2.4)** 3.0 0.8 1.2 0.4** 1.27 0.25** 0.98 0.24* 127.5 (83.8e170.4)**
Data are expressed as mean SD or median (interquartile range). *p < 0.05 and **p < 0.01 versus healthy controls.
inflammatory response. For example, there is epidemiological data linking SAA with cardiovascular disease [24,25], and it has also been shown that SAA may be proatherogenic [26]. In our study, SAA is a significant determinant of SR-BI-mediated cholesterol efflux to serum, and our experimental data suggest that this is unlikely to be related to HDL enrichment with SAA. In acute phase HDL, SAA may account for up to 17%e87% of the total apolipoproteins present in HDL [27], and Banka et al. reported that the cholesterol efflux property of HDL was only reduced when SAA constituted more than 50% of HDL protein [5]. The level of SAA in serum was much lower in our diabetic subjects than that seen in acute phase response. Even in subjects in the highest quartile of SAA in serum, HDL-SAA accounted for less than 5% of HDL protein. Hence, the degree of SAA enrichment of HDL was not sufficient to alter the ability of HDL to act as cholesterol acceptor and unlikely to
Fig. 1. SR-BI-mediated cholesterol efflux to serum (1a) and ABCG1-mediated cholesterol efflux to serum (1b) in controls and diabetic subjects. Data are expressed as mean SD. *p < 0.05 compared with healthy controls.
account for the impairment in SR-BI-mediated cholesterol efflux to serum. Lipid-free SAA is not a cholesterol acceptor for SR-BI [28], but can act as a ligand to SR-BI [16]. Results from our binding assay suggest that lipid-free SAA can inhibit the binding of HDL to SR-BI and is similar to the findings of Cai et al. [16]. Since SR-BI-mediated cholesterol efflux from cells to HDL requires the binding of HDL to SR-BI particularly at low acceptor concentration [29], inhibition of
Fig. 2. Correlation between SAA and SR-BI-mediated cholesterol efflux to serum, r ¼ 0.36, p < 0.01 (2a), and ABCG1-mediated cholesterol efflux to serum (2b).
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In conclusion, SAA was increased in type 2 diabetic patients with incipient or overt nephropathy, and SAA was associated with impairment of SR-BI-mediated cholesterol efflux to serum. Financial support This study is supported by funding from the Hong Kong Research Grants Council Research Fund [HKU 776709M]. Conflict of interest The authors have no conflict of interest to disclose. References
Fig. 3. Correlation between SAA and HDL-SAA in diabetic subjects, r ¼ 0.49, p < 0.05 (3a). Effect of lipid-free human SAA on HDL binding to SR-BI in Fu5AH cells (3b).
HDL binding by lipid-free SAA may lead to impairment of cholesterol efflux. In addition, SAA has an inhibitory effect on LCAT activity [30], which may also lead to impairment in cholesterol efflux to serum [18]. We did not find any association between SAA and ABCG1-mediated cholesterol efflux and this is in keeping with experimental data showing that ABCG1-dependent cholesterol efflux was independent of SAA during inflammation [31]. Our study has several limitations. The control subjects were significantly younger than the diabetic patients, and we have adjusted for age as a confounding factor in our analysis. Because of its cross-sectional design, we can only demonstrate associations and not causal relationships. We have only investigated the effect of SAA on cholesterol efflux capacity of serum which reflects the ability to mobilise free cholesterol from cells. Although it has been shown that cholesterol efflux capacity is associated with atherosclerosis in humans [19], we have not evaluated other components of the reverse cholesterol pathway. Another major limitation is that we have not included ABCA1-mediated cholesterol efflux to serum in the present study, and we cannot comment on the overall effect of SAA on cholesterol efflux derangement in type 2 diabetic patients. Work is on-going and our preliminary data on ABCA1mediated cholesterol efflux to serum have so far shown that ABCA1-mediated cholesterol efflux to serum was significantly impaired in diabetic patients with proteinuria (unpublished data) which was in keeping with our earlier study [12].
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