Impact of serum amyloid A on cellular cholesterol efflux to serum in type 2 diabetes mellitus.

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 [(3)H]cholesterol from Fu5AH rat hepatoma cells expressing SR-BI and from human ABCG1-transfected 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 = -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.

Effect of insulin on the soluble receptor for advanced glycation end products (RAGE).

AIMS: The receptor for advanced glycation end products (RAGE) plays an important role in the pathogenesis of diabetic complications. RAGE transcript splicing generates a number of isoforms, including a full-length membrane-bound receptor and a soluble isoform, endogenous secretory RAGE (esRAGE). Soluble forms of the receptor (sRAGE) can also be formed by ectodomain shedding of the membrane-associated receptor. We have evaluated serum levels of sRAGE and esRAGE in Chinese patients with Type 1 diabetes and investigated the effect of insulin on the generation of esRAGE and sRAGE in vitro. METHODS: Serum sRAGE and esRAGE were measured by ELISA. The in vitro effect of insulin was investigated by incubating THP-1 macrophages with insulin and RAGE isoforms in cell lysate and conditioned media determined. RESULTS: In patients with diabetes, both serum esRAGE and sRAGE were significantly higher than in age-matched healthy subjects without diabetes. In vitro, insulin increased esRAGE and total RAGE isoform expression in cell lysate on a western blot, and reverse transcription-polymerase chain reaction showed an increase in esRAGE and full-length RAGE mRNA. This was accompanied by an increase in esRAGE and sRAGE in cell conditioned media. Pretreatment of THP-1 cells with a general metalloproteinase inhibitor GM6001 significantly reduced the production of sRAGE, suggesting that insulin also increased the cleavage of full-length cell surface RAGE to form sRAGE. CONCLUSIONS: Chinese patients with Type 1 diabetes have higher serum levels of esRAGE and sRAGE. In vitro, insulin not only increases both full-length RAGE and esRAGE expression, but can also stimulate the shedding of sRAGE from the membrane-bound receptor.

Effects of atorvastatin on serum soluble receptors for advanced glycation end-products in type 2 diabetes.

OBJECTIVE: The receptor for advanced glycation end-products (RAGE) plays an important role in the pathogenesis of diabetic complications and atherosclerosis. Interfering with the activation of RAGE by using a soluble form of the receptor (sRAGE) ameliorates the vascular complications of diabetes in animal models. We have investigated whether statin can influence the expression of sRAGE and esRAGE (a splice variant of sRAGE) in vitro and in vivo. METHODS: THP-1 cells were incubated with atorvastatin in vitro and sRAGE and esRAGE in the medium was measured by Western immunoblot. Serum levels of sRAGE and esRAGE were measured by ELISA in archived serum samples from a previous randomized double-blind placebo-controlled clinical trial that explored the cardiovascular effects of atorvastatin in hypercholesterolemic Chinese type 2 diabetic patients. RESULTS: sRAGE and esRAGE were induced by atorvastatin in a time- and dose-dependent manner in THP-1 cells. In the diabetic patients, there was a significant increase in serum sRAGE (p<0.05) and esRAGE (p<0.01) in the atorvastatin group at 6-month, but no change in placebo group. Serum esRAGE was higher in atorvastatin group than placebo group [median 240.5pg/ml (interquartile range 186.5-377.3) vs 194.8pg/ml (124.1-347.9) respectively, p<0.01] at 6-month, whereas the differences in sRAGE did not reach statistical significance (p=0.051). There was a correlation between the increase of serum esRAGE and reduction of serum LDL (r=-0.36, p=0.001). CONCLUSIONS: Statins are known to have pleiotropic effects and we have shown that atorvastatin can increase circulating esRAGE levels in type 2 diabetic patients.

Association between serum levels of soluble receptor for advanced glycation end products and circulating advanced glycation end products in type 2 diabetes.

AIMS/HYPOTHESIS: Activation of the receptor for advanced glycation end products (RAGE, also known as AGE-specific receptor [AGER]) has been implicated in the development of diabetic vascular complications. Blockade of RAGE using a soluble form of the receptor (sRAGE) suppressed vascular hyperpermeability and atherosclerosis in animal models. Since little is known about the regulation of endogenous sRAGE levels, we determined whether serum sRAGE is influenced by circulating AGEs and the severity of nephropathy in type 2 diabetic patients. MATERIALS AND METHODS: We recruited 150 healthy control and 318 diabetic subjects. Diabetic subjects were subdivided into those with proteinuria, microalbuminuria or normoalbuminuria. Serum sRAGE was assayed by ELISA and serum AGEs by competitive ELISA using a polyclonal rabbit antiserum raised against AGE-RNase. RESULTS: Diabetic subjects had higher sRAGE (1,029.5 pg/ml [766.1-1,423.0] interquartile range vs 1,002.6 [726.5-1,345.3], p<0.05) and AGEs (4.07+/-1.13, SD, unit/ml vs 3.39+/-1.05, p<0.01) than controls. Proteinuric subjects had the highest sRAGE levels and there was a significant trend between the severity of nephropathy and sRAGE (p=0.01). In diabetic subjects, serum log(sRAGE) correlated with AGEs (r=0.27, p<0.001), log(plasma creatinine) (r=0.31, p<0.001), log(urine AER) (r=0.24, p<0.01) and log(triglycerides) (r=0.15, p<0.01). On stepwise linear regression analysis, AGEs and creatinine levels were the main independent determinants of sRAGE concentration. CONCLUSIONS/INTERPRETATION: Serum sRAGE levels and circulating AGEs are associated with the severity of nephropathy in type 2 diabetic patients. Prospective studies are required to determine whether endogenous sRAGE potentially influences the development of diabetic vascular complications.

Plasma apolipoprotein E concentration is an important determinant of phospholipid transfer protein activity in type 2 diabetes mellitus.

BACKGROUND: Phospholipid transfer protein (PLTP) transfers phospholipids between lipoproteins and plays an important role in HDL metabolism. PLTP exists as a high-activity and a low-activity form in the circulation. In vitro studies have shown that apolipoprotein (apo) E is involved in maintaining PLTP in the active form, while the low-activity form is associated with apo AI. We have therefore investigated whether plasma apo AI, B and E concentrations are important determinants of plasma PLTP activity in type 2 diabetes, a condition associated with increased plasma PLTP activity. METHODS: Plasma PLTP activity was assayed by measuring the transfer of radiolabelled phosphatidylcholine from liposomes to HDL; apo AI and B by rate nephelometry and apo E by a 2-point turbidimetric assay. RESULTS: Type 2 diabetic patients (n = 230) had higher PLTP activity than controls (n = 97) (2374 +/- 628 nmol/mL/h versus 1862 +/- 585 respectively, p < 0.01). They also had increased fasting triglyceride and low HDL. Plasma apo B (p < 0.01) and apo E (p < 0.05) were increased, whereas apo AI was reduced (p < 0.01). Univariate analysis showed that plasma PLTP activity correlated mainly with apolipoproteins AI and E. Stepwise regression analysis showed that apo E was the main determinant of plasma PLTP activity, accounting for 23% of its variability in the diabetic subjects and 8% in the controls respectively. CONCLUSIONS: The associations between plasma apo AI and E concentrations and PLTP activity suggest that these apolipoproteins are important regulators of PLTP activity in vivo. The increase in PLTP activity in type 2 diabetes is partly related to the changes in these apolipoproteins.

Plasma phospholipid transfer protein activity and subclinical inflammation in type 2 diabetes mellitus.

Phospholipid transfer protein (PLTP) transfers phospholipids between lipoproteins, and plays an essential role in HDL metabolism. The regulation of PLTP is poorly understood and recent evidence suggests that PLTP activity increases during acute-phase response. Since type 2 diabetes is associated with chronic subclinical inflammation, the objective is to determine whether inflammation modulates PLTP in diabetes. Plasma PLTP activity was assayed by measuring the transfer of radiolabeled phosphatidylcholine from liposomes to HDL and high-sensitivity C-reactive protein (CRP) by immunoturbidimetric assay in 280 type 2 diabetic patients and 105 controls. Plasma PLTP activity (2364+/-651 nmol/ml/h versus 1880+/-586 nmol/ml/h in control, mean +/- S.D., P <0.01) and CRP (1.64(0.89-3.23)mg/l versus 0.99(0.53-2.23 mg/l, median (interquartile range), P<0.01) were increased in diabetic subjects. PLTP activity correlated significantly with age, BMI, HbA1c, log(CRP) and apolipoprotein AI and B in diabetic subjects. General linear model analysis showed that only apolipoprotein AI, age, BMI, and log(CRP) were independent determinants of PLTP activity. In conclusion, PLTP activity is increased in diabetes and apolipoprotein AI is a major determinant of PLTP activity. There is also an independent association between CRP and PLTP activity, suggesting that subclinical inflammation may influence PLTP activity in diabetes.

Plasma phospholipid transfer protein activity and small, dense LDL in type 2 diabetes mellitus.

BACKGROUND: Phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) remodel circulating lipoproteins and play a role in the antiatherogenic reverse cholesterol transport pathway. The present study determined whether abnormalities in the LDL subfraction pattern in type 2 diabetic patients were related to changes in lipid transfer proteins. METHODS: Low-density lipoprotein (LDL) subfractions were measured by density gradient ultracentrifugation and plasma PLTP and CETP activities by radiometric assays in 240 diabetic patients and 136 controls. RESULTS: The diabetic patients had lower LDL-I (P < 0.001) and higher LDL-III concentrations than the controls (P < 0.001). Plasma PLTP activity was increased (P < 0.001) whereas no significant differences were seen in CETP activity. In the diabetic patients, small, dense LDL-III correlated with plasma triglyceride (r = 0.18, P < 0.01), HDL (r = -0.14, P < 0.05), PLTP (r = 0.29, P < 0.001) and CETP activity (r = 0.15, P < 0.05). Linear regression analysis showed that plasma PLTP activity, triglyceride and age were the major determinants of LDL-III concentration (r2 = 28%, P < 0.001). The univariate relationship between CETP and LDL-III was no longer significant after adjusting for PLTP activity. CONCLUSIONS: The increase in plasma PLTP activity was independently associated with small, dense LDL concentrations in type 2 diabetes. Hence, elevated PLTP activity might have both antiatherogenic and pro-atherogenic potential in these patients.