FIG 3 - uploaded by Yoshinari Uehara
Content may be subject to copyright.
Interacting effects of 22( R )-hydroxycholesterol and 9- cis- RA with fatty acids and ketone bodies on the expression of ABCA1 in HepG2 cells and RAW264.7 cells. Con fl uent hepatocytes ( A and C ) and RAW264.7 cells ( B and D ) were incubated for 18 h with serum-free medium in the presence of 10 g/l BSA and 10 mol/l 22R-HC ( A and B ) or 10 mol/l 9- cis- RA ( C and D ) plus 100 mol/l EPA, 500 mol/l palmitic acid (PA), 500 mol/l oleic acid (OA), 10 mmol/l hydroxybutyrate (HB), or 10 mmol/l acetoacetate (AA). The mRNA of ABCA1 was demonstrated by quantita- tive real-time PCR. Note that EPA, oleic acid, and acetoacetate (more prominent in macrophages, but not palmitic acid and hydroxybutyrate) ( B ) suppress the stimulatory effect of 22( R )-HC on ABCA1 gene expression. EPA and oleic acid have less suppression on ABCA1 expression in the presence of 9- cis- RA than in 22( R )-HC. Results represent means ؎ SD from four to six experiments. † P < 0.01, * P < 0.05 compared with control (one-way ANOVA).
Source publication
Low HDL cholesterol is a frequent cardiovascular risk factor in diabetes. Because of its pivotal role for the regulation of HDL plasma levels, we investigated in vivo and in vitro regulation of the ATP-binding cassette transporter A1 (ABCA1) by insulin and metabolites accumulating in diabetes. Compared with euglycemic control mice, ABCA1 gene expre...
Contexts in source publication
Context 1
... clinical and epidemiological studies have demonstrated the inverse association between HDL cholesterol and the risk of coronary heart disease events (1). It is controversially discussed whether this relationship is causal or only an epiphenomenon of a more general atherogenic disorder. On the one hand, HDL exerts various potentially anti- atherogenic properties. For example, HDL particles transport cholesterol from cells of the arterial wall to the liver and to steroidogenic organs, in which cholesterol is used for the synthesis of bile acids, lipoproteins, vitamin D, and steroid hormones (1). On the other hand, low HDL cholesterol is frequently found as a component of the metabolic syndrome in many populations, i.e., together with overweight or obesity, glucose intolerance or overt diabetes, hypertriglyceridemia, and hypertension, which by themselves contribute to the pathogenesis of atherosclerosis (2). Moreover, many individuals with low HDL cholesterol have elevated fasting plasma levels of insulin, are resistant to exogenous insulin in euglycemic clamp studies, and bear an increased risk for future type 2 diabetes (3–5). The pathogenesis of low HDL cholesterol in insulin resistance is not well understood but may provide an important key to answer the question of whether low HDL cholesterol is a causal factor in the pathogenesis of atherosclerosis in patients with insulin resistance or diabetes. ATP-binding cassette transporter A1 (ABCA1) has previously been identified as a pivotal gene in the regulation of both HDL cholesterol plasma levels and the cellular cholesterol homeostasis, which is defective in patients with Tangier disease (1,6 –9). In these patients and their heterozygous relatives, mutations in the ABCA1 gene cause gene dosage– dependent decreases in plasma levels of HDL cholesterol and in the capacity of skin fibroblasts and monocyte-derived macrophages to release cholesterol in the extracellular presence of apolipoproteins (1,6 –9). As the clinical result, homozygous patients with Tangier disease accumulate macrophage-derived foam cells in various tissues and develop tonsil anomalies, hepato- splenomegaly, premature atherosclerosis, and peripheral neuropathy (10). Cyclic AMP and ligands of the nuclear transcription factors liver-X-receptor ␣ (LXR ␣ ) and retinoid-X-receptor (RXR ), i.e., oxysterols and retinoids, respectively, have been identi fi ed as enhancers of ABCA1 gene expression (11 – 14). By contrast, interferon- ␥ (IFN ␥ ) downregulates ABCA1 gene expression (15). Here we investigated the regulation of ABCA1 in a mouse model of diabetes. Since ABCA1 gene expression was substantially decreased in the liver and peritoneal macrophages of diabetic mice, we next investigated the effects of insulin as well as metabolites accumulating in diabetes on ABCA1 gene expression. Thereby, we identi fi ed free polyunsaturated fatty acids (PUFAs) and acetoacetate as strong suppressors of ABCA1 gene expression. To test the possible contribution of dysregulated ABCA1 gene expression to low HDL cholesterol, we initially investigated the regulation of ABCA1 in mice that were made diabetic with streptozotocin (Table 2, Fig. 1). Compared with untreated mice, the liver (Fig. 1 A ) and peritoneal macrophages (Fig. 1 B ) of diabetic mice showed a marked decrease in the expression of ABCA1 mRNA. Intraperitoneal injection of insulin abolished the diabetic phenotype (Table 2) and restored the expression of ABCA1 in either liver (Fig. 1 C ) or peritoneal macrophages (Fig. 1 D ). Our next experiments were aimed at the identi fi cation of the metabolic basis of the decreased ABCA1 gene expression in diabetes. We incubated HepG2 hepatocytes (Fig. 2 A ) or RAW264.7 macrophages (Fig. 2 B ) for 18 h with increasing dosages of insulin, glucose, fatty acids, or ketone bodies. Addition of insulin or glucose into the cell culture medium had no consistent effects on the expression of ABCA1 in either hepatocytes or macrophages. By contrast, the polyunsaturated EPA as well as the ketone body acetoacetate dose-dependently decreased the expression of ABCA1 in both cell lines (Figs. 2 A and B ). The suppressive effects of these fatty acids became most evident in those cells where ABCA1 expression was up- regulated with an oxysterol, i.e., an activator of the nuclear transcription factor LXR ␣ , which was previously shown to induce the ABCA1 gene (12,13). In HepG2 cells, the stimulatory effect of 22R-HC (12,13) on ABCA1 was inhibited by ϳ 80% ( P Ͻ 0.01), 60% ( P Ͻ 0.01), and 40% ( P Ͻ 0.05) in the additional presence of physiological dosages of EPA (100 mol/l), oleic acid (500 mol/l), or acetotac- etate (10 mmol/l), respectively (Fig. 3 A ). In RAW264.7 cells, the respective numbers were 95, 80, and 60% (all P Ͻ 0.01; Fig. 3 B ). Also, in the presence of 9- cis- RA, i.e., an activator of the nuclear transcription factor RXR ␣ that forms heterodimers with LXR ␣ and thereby also induces the ABCA1 gene (14), EPA, oleic acid, and acetoacetate suppressed ABCA1 gene expression in HepG2 and RAW by 50 – 60% (Figs. 3 C and D , all signi fi cant at a level of P Ͻ 0.05 or P Ͻ 0.01, except acetoacetate in HepG2 cells). In none of these conditions did palmitic acid or hydroxybutyrate modulate ABCA1 gene expression (Fig. 3 A – D ). Likewise, and in agreement with data from Wang and Oram (19), we did not see any suppressive effect of fatty acids or acetoacetate on ABCA1 gene expression in macrophages that were stimulated with Br-cAMP (data not shown). We veri fi ed the stimulatory effects of 22(R)-HC and 9- cis- RA as well as the inhibitory effects of EPA and acetoacetate in a luciferase reporter gene assay, which was performed on a construct that contained 0.968 kb of the human ABCA1 promotor (Fig. 4). As expected from previous reports (12 – 14), 22R-HC and 9- cis- RA increased the ABCA1 promotor activity by a factor up to 30 (Figs. 4 A and B ). EPA and acetoacetate suppressed these stimula- tory effects dose dependently by up to 80% (EPA, Figs. 4 C and D ) and 50% (acetoacetate, Figs. 4 E and F ), respectively. Using this reporter gene assay, we compared the suppressive effects of various fatty acid species at a concentration of 100 mol/l. Linoleic acid was most effec- tive ( Ϫ 80%), followed by arachidonic acid ( Ϫ 75%), EPA ( Ϫ 70%), and oleic acid ( Ϫ 40%). Palmitic acid had a modest but signi fi cant suppressive effect ( Ϫ 10%), whereas stearic acid exerted a moderate but also signi fi cant stimulatory effect ( ϩ 10%, Fig. 5). Our observations raised the question as to whether unsaturated fatty acids and acetoacetate regulate the expression of ABCA1 indirectly via regulation of LXR ␣ or RXR ␣ gene expression. Such a mechanism was previously demonstrated to be responsible for the upregulation of ABCA1 by agonists of peroxisome proliferator – activated receptor (PPAR)- ␣ (i.e., fi brates) and PPAR ␥ (i.e., glita- zones) (20). Neither oleic acid nor EPA nor acetoacetate regulated the expression of LXR ␣ or RXR ␣ (data not shown). Immunoprecipitation and subsequent Western blot analysis clearly detected ABCA1 in RAW264.7 cells (Fig. 6 A ), which were cultivated in the presence of an oxysterol. However, the cellular ABCA1 protein concentration was severely decreased when these cells were incubated in the additional presence of free fatty acids or acetoacetate (Fig. 6 A ). The functional relevance of ABCA1 downregulation by free fatty acids and ketone bodies was investigated in cholesterol ef fl ux experiments (Fig. 6 B ). Lipid- free apoA-I induced signi fi cant cholesterol ef fl ux from RAW264.7 cells. As reported previously, cholesterol ef fl ux to apoA-I was stimulated by oxysterols (11 – 13). The stimulatory effect of oxysterols was completely inhibited by 500 mol/l oleic acid, 100 mol/l EPA, and 10 mmol/l acetoacetate (Fig. 6 B ). Interestingly, the expression of another important gene involved in cholesterol ef fl ux, scavenger receptor BI, was not affected by either fatty acids or acetoacetate (data not shown). Here we have demonstrated that ABCA1 is tremendously downregulated in an animal model of diabetes (Fig. 1). As the most likely explanation, we found suppressive effects of PUFAs, oleic acid, and acetoacetate on ABCA1 gene expression in vitro (Figs. 2 – 5). Interestingly, the saturated palmitic and stearic acids as well as the ketone body 3-hydroxybutyrate did not alter ABCA1 gene expression so that the inhibitory effects appear to be speci fi c for unsaturated fatty acids and acetoacetate. Suppression of ABCA1 gene expression by unsaturated fatty acids became most obvious in the additional presence of an oxysterol (Fig. 3) but did not occur in the presence of Br-cAMP (data not shown, 19). This raised the possibility that unsaturated fatty acids downregulate ABCA1 via suppression or inhibition of the nuclear transcription factor LXR ␣ , which is activated by oxysterols and which upregulates ABCA1 (12,13,21). Free fatty acids were previously shown to interact with LXR ␣ on both the transcriptional and the posttranslational level (22,23). Free fatty acids induce the transcription of the LXR gene via activation of PPARs (22,24). However, unsaturated fatty acids do not suppress ABCA1 via transcriptional regulation of LXR ␣ , since in our experiments oleic acid and EPA did not modulate the expression of LXR ␣ (data not shown) and since PPAR ␣ , PPAR ␥ , and PPAR ␦ rather upregulate ABCA1 via stimulation of LXR ␣ transcription (14,25). On the posttranslational level, unsaturated fatty acids antag- onize the activation of LXR ␣ by oxysterols and thereby inhibit the transcription of the sterol regulatory element- binding protein-1c (23,26). This posttranslational inhibition of LXR ␣ well explains the transcriptional ...
Context 2
... clinical and epidemiological studies have demonstrated the inverse association between HDL cholesterol and the risk of coronary heart disease events (1). It is controversially discussed whether this relationship is causal or only an epiphenomenon of a more general atherogenic disorder. On the one hand, HDL exerts various potentially anti- atherogenic properties. For example, HDL particles transport cholesterol from cells of the arterial wall to the liver and to steroidogenic organs, in which cholesterol is used for the synthesis of bile acids, lipoproteins, vitamin D, and steroid hormones (1). On the other hand, low HDL cholesterol is frequently found as a component of the metabolic syndrome in many populations, i.e., together with overweight or obesity, glucose intolerance or overt diabetes, hypertriglyceridemia, and hypertension, which by themselves contribute to the pathogenesis of atherosclerosis (2). Moreover, many individuals with low HDL cholesterol have elevated fasting plasma levels of insulin, are resistant to exogenous insulin in euglycemic clamp studies, and bear an increased risk for future type 2 diabetes (3–5). The pathogenesis of low HDL cholesterol in insulin resistance is not well understood but may provide an important key to answer the question of whether low HDL cholesterol is a causal factor in the pathogenesis of atherosclerosis in patients with insulin resistance or diabetes. ATP-binding cassette transporter A1 (ABCA1) has previously been identified as a pivotal gene in the regulation of both HDL cholesterol plasma levels and the cellular cholesterol homeostasis, which is defective in patients with Tangier disease (1,6 –9). In these patients and their heterozygous relatives, mutations in the ABCA1 gene cause gene dosage– dependent decreases in plasma levels of HDL cholesterol and in the capacity of skin fibroblasts and monocyte-derived macrophages to release cholesterol in the extracellular presence of apolipoproteins (1,6 –9). As the clinical result, homozygous patients with Tangier disease accumulate macrophage-derived foam cells in various tissues and develop tonsil anomalies, hepato- splenomegaly, premature atherosclerosis, and peripheral neuropathy (10). Cyclic AMP and ligands of the nuclear transcription factors liver-X-receptor ␣ (LXR ␣ ) and retinoid-X-receptor (RXR ), i.e., oxysterols and retinoids, respectively, have been identi fi ed as enhancers of ABCA1 gene expression (11 – 14). By contrast, interferon- ␥ (IFN ␥ ) downregulates ABCA1 gene expression (15). Here we investigated the regulation of ABCA1 in a mouse model of diabetes. Since ABCA1 gene expression was substantially decreased in the liver and peritoneal macrophages of diabetic mice, we next investigated the effects of insulin as well as metabolites accumulating in diabetes on ABCA1 gene expression. Thereby, we identi fi ed free polyunsaturated fatty acids (PUFAs) and acetoacetate as strong suppressors of ABCA1 gene expression. To test the possible contribution of dysregulated ABCA1 gene expression to low HDL cholesterol, we initially investigated the regulation of ABCA1 in mice that were made diabetic with streptozotocin (Table 2, Fig. 1). Compared with untreated mice, the liver (Fig. 1 A ) and peritoneal macrophages (Fig. 1 B ) of diabetic mice showed a marked decrease in the expression of ABCA1 mRNA. Intraperitoneal injection of insulin abolished the diabetic phenotype (Table 2) and restored the expression of ABCA1 in either liver (Fig. 1 C ) or peritoneal macrophages (Fig. 1 D ). Our next experiments were aimed at the identi fi cation of the metabolic basis of the decreased ABCA1 gene expression in diabetes. We incubated HepG2 hepatocytes (Fig. 2 A ) or RAW264.7 macrophages (Fig. 2 B ) for 18 h with increasing dosages of insulin, glucose, fatty acids, or ketone bodies. Addition of insulin or glucose into the cell culture medium had no consistent effects on the expression of ABCA1 in either hepatocytes or macrophages. By contrast, the polyunsaturated EPA as well as the ketone body acetoacetate dose-dependently decreased the expression of ABCA1 in both cell lines (Figs. 2 A and B ). The suppressive effects of these fatty acids became most evident in those cells where ABCA1 expression was up- regulated with an oxysterol, i.e., an activator of the nuclear transcription factor LXR ␣ , which was previously shown to induce the ABCA1 gene (12,13). In HepG2 cells, the stimulatory effect of 22R-HC (12,13) on ABCA1 was inhibited by ϳ 80% ( P Ͻ 0.01), 60% ( P Ͻ 0.01), and 40% ( P Ͻ 0.05) in the additional presence of physiological dosages of EPA (100 mol/l), oleic acid (500 mol/l), or acetotac- etate (10 mmol/l), respectively (Fig. 3 A ). In RAW264.7 cells, the respective numbers were 95, 80, and 60% (all P Ͻ 0.01; Fig. 3 B ). Also, in the presence of 9- cis- RA, i.e., an activator of the nuclear transcription factor RXR ␣ that forms heterodimers with LXR ␣ and thereby also induces the ABCA1 gene (14), EPA, oleic acid, and acetoacetate suppressed ABCA1 gene expression in HepG2 and RAW by 50 – 60% (Figs. 3 C and D , all signi fi cant at a level of P Ͻ 0.05 or P Ͻ 0.01, except acetoacetate in HepG2 cells). In none of these conditions did palmitic acid or hydroxybutyrate modulate ABCA1 gene expression (Fig. 3 A – D ). Likewise, and in agreement with data from Wang and Oram (19), we did not see any suppressive effect of fatty acids or acetoacetate on ABCA1 gene expression in macrophages that were stimulated with Br-cAMP (data not shown). We veri fi ed the stimulatory effects of 22(R)-HC and 9- cis- RA as well as the inhibitory effects of EPA and acetoacetate in a luciferase reporter gene assay, which was performed on a construct that contained 0.968 kb of the human ABCA1 promotor (Fig. 4). As expected from previous reports (12 – 14), 22R-HC and 9- cis- RA increased the ABCA1 promotor activity by a factor up to 30 (Figs. 4 A and B ). EPA and acetoacetate suppressed these stimula- tory effects dose dependently by up to 80% (EPA, Figs. 4 C and D ) and 50% (acetoacetate, Figs. 4 E and F ), respectively. Using this reporter gene assay, we compared the suppressive effects of various fatty acid species at a concentration of 100 mol/l. Linoleic acid was most effec- tive ( Ϫ 80%), followed by arachidonic acid ( Ϫ 75%), EPA ( Ϫ 70%), and oleic acid ( Ϫ 40%). Palmitic acid had a modest but signi fi cant suppressive effect ( Ϫ 10%), whereas stearic acid exerted a moderate but also signi fi cant stimulatory effect ( ϩ 10%, Fig. 5). Our observations raised the question as to whether unsaturated fatty acids and acetoacetate regulate the expression of ABCA1 indirectly via regulation of LXR ␣ or RXR ␣ gene expression. Such a mechanism was previously demonstrated to be responsible for the upregulation of ABCA1 by agonists of peroxisome proliferator – activated receptor (PPAR)- ␣ (i.e., fi brates) and PPAR ␥ (i.e., glita- zones) (20). Neither oleic acid nor EPA nor acetoacetate regulated the expression of LXR ␣ or RXR ␣ (data not shown). Immunoprecipitation and subsequent Western blot analysis clearly detected ABCA1 in RAW264.7 cells (Fig. 6 A ), which were cultivated in the presence of an oxysterol. However, the cellular ABCA1 protein concentration was severely decreased when these cells were incubated in the additional presence of free fatty acids or acetoacetate (Fig. 6 A ). The functional relevance of ABCA1 downregulation by free fatty acids and ketone bodies was investigated in cholesterol ef fl ux experiments (Fig. 6 B ). Lipid- free apoA-I induced signi fi cant cholesterol ef fl ux from RAW264.7 cells. As reported previously, cholesterol ef fl ux to apoA-I was stimulated by oxysterols (11 – 13). The stimulatory effect of oxysterols was completely inhibited by 500 mol/l oleic acid, 100 mol/l EPA, and 10 mmol/l acetoacetate (Fig. 6 B ). Interestingly, the expression of another important gene involved in cholesterol ef fl ux, scavenger receptor BI, was not affected by either fatty acids or acetoacetate (data not shown). Here we have demonstrated that ABCA1 is tremendously downregulated in an animal model of diabetes (Fig. 1). As the most likely explanation, we found suppressive effects of PUFAs, oleic acid, and acetoacetate on ABCA1 gene expression in vitro (Figs. 2 – 5). Interestingly, the saturated palmitic and stearic acids as well as the ketone body 3-hydroxybutyrate did not alter ABCA1 gene expression so that the inhibitory effects appear to be speci fi c for unsaturated fatty acids and acetoacetate. Suppression of ABCA1 gene expression by unsaturated fatty acids became most obvious in the additional presence of an oxysterol (Fig. 3) but did not occur in the presence of Br-cAMP (data not shown, 19). This raised the possibility that unsaturated fatty acids downregulate ABCA1 via suppression or inhibition of the nuclear transcription factor LXR ␣ , which is activated by oxysterols and which upregulates ABCA1 (12,13,21). Free fatty acids were previously shown to interact with LXR ␣ on both the transcriptional and the posttranslational level (22,23). Free fatty acids induce the transcription of the LXR gene via activation of PPARs (22,24). However, unsaturated fatty acids do not suppress ABCA1 via transcriptional regulation of LXR ␣ , since in our experiments oleic acid and EPA did not modulate the expression of LXR ␣ (data not shown) and since PPAR ␣ , PPAR ␥ , and PPAR ␦ rather upregulate ABCA1 via stimulation of LXR ␣ transcription (14,25). On the posttranslational level, unsaturated fatty acids antag- onize the activation of LXR ␣ by oxysterols and thereby inhibit the transcription of the sterol regulatory element- binding protein-1c ...
Context 3
... one hand, HDL exerts various potentially anti- atherogenic properties. For example, HDL particles transport cholesterol from cells of the arterial wall to the liver and to steroidogenic organs, in which cholesterol is used for the synthesis of bile acids, lipoproteins, vitamin D, and steroid hormones (1). On the other hand, low HDL cholesterol is frequently found as a component of the metabolic syndrome in many populations, i.e., together with overweight or obesity, glucose intolerance or overt diabetes, hypertriglyceridemia, and hypertension, which by themselves contribute to the pathogenesis of atherosclerosis (2). Moreover, many individuals with low HDL cholesterol have elevated fasting plasma levels of insulin, are resistant to exogenous insulin in euglycemic clamp studies, and bear an increased risk for future type 2 diabetes (3–5). The pathogenesis of low HDL cholesterol in insulin resistance is not well understood but may provide an important key to answer the question of whether low HDL cholesterol is a causal factor in the pathogenesis of atherosclerosis in patients with insulin resistance or diabetes. ATP-binding cassette transporter A1 (ABCA1) has previously been identified as a pivotal gene in the regulation of both HDL cholesterol plasma levels and the cellular cholesterol homeostasis, which is defective in patients with Tangier disease (1,6 –9). In these patients and their heterozygous relatives, mutations in the ABCA1 gene cause gene dosage– dependent decreases in plasma levels of HDL cholesterol and in the capacity of skin fibroblasts and monocyte-derived macrophages to release cholesterol in the extracellular presence of apolipoproteins (1,6 –9). As the clinical result, homozygous patients with Tangier disease accumulate macrophage-derived foam cells in various tissues and develop tonsil anomalies, hepato- splenomegaly, premature atherosclerosis, and peripheral neuropathy (10). Cyclic AMP and ligands of the nuclear transcription factors liver-X-receptor ␣ (LXR ␣ ) and retinoid-X-receptor (RXR ), i.e., oxysterols and retinoids, respectively, have been identi fi ed as enhancers of ABCA1 gene expression (11 – 14). By contrast, interferon- ␥ (IFN ␥ ) downregulates ABCA1 gene expression (15). Here we investigated the regulation of ABCA1 in a mouse model of diabetes. Since ABCA1 gene expression was substantially decreased in the liver and peritoneal macrophages of diabetic mice, we next investigated the effects of insulin as well as metabolites accumulating in diabetes on ABCA1 gene expression. Thereby, we identi fi ed free polyunsaturated fatty acids (PUFAs) and acetoacetate as strong suppressors of ABCA1 gene expression. To test the possible contribution of dysregulated ABCA1 gene expression to low HDL cholesterol, we initially investigated the regulation of ABCA1 in mice that were made diabetic with streptozotocin (Table 2, Fig. 1). Compared with untreated mice, the liver (Fig. 1 A ) and peritoneal macrophages (Fig. 1 B ) of diabetic mice showed a marked decrease in the expression of ABCA1 mRNA. Intraperitoneal injection of insulin abolished the diabetic phenotype (Table 2) and restored the expression of ABCA1 in either liver (Fig. 1 C ) or peritoneal macrophages (Fig. 1 D ). Our next experiments were aimed at the identi fi cation of the metabolic basis of the decreased ABCA1 gene expression in diabetes. We incubated HepG2 hepatocytes (Fig. 2 A ) or RAW264.7 macrophages (Fig. 2 B ) for 18 h with increasing dosages of insulin, glucose, fatty acids, or ketone bodies. Addition of insulin or glucose into the cell culture medium had no consistent effects on the expression of ABCA1 in either hepatocytes or macrophages. By contrast, the polyunsaturated EPA as well as the ketone body acetoacetate dose-dependently decreased the expression of ABCA1 in both cell lines (Figs. 2 A and B ). The suppressive effects of these fatty acids became most evident in those cells where ABCA1 expression was up- regulated with an oxysterol, i.e., an activator of the nuclear transcription factor LXR ␣ , which was previously shown to induce the ABCA1 gene (12,13). In HepG2 cells, the stimulatory effect of 22R-HC (12,13) on ABCA1 was inhibited by ϳ 80% ( P Ͻ 0.01), 60% ( P Ͻ 0.01), and 40% ( P Ͻ 0.05) in the additional presence of physiological dosages of EPA (100 mol/l), oleic acid (500 mol/l), or acetotac- etate (10 mmol/l), respectively (Fig. 3 A ). In RAW264.7 cells, the respective numbers were 95, 80, and 60% (all P Ͻ 0.01; Fig. 3 B ). Also, in the presence of 9- cis- RA, i.e., an activator of the nuclear transcription factor RXR ␣ that forms heterodimers with LXR ␣ and thereby also induces the ABCA1 gene (14), EPA, oleic acid, and acetoacetate suppressed ABCA1 gene expression in HepG2 and RAW by 50 – 60% (Figs. 3 C and D , all signi fi cant at a level of P Ͻ 0.05 or P Ͻ 0.01, except acetoacetate in HepG2 cells). In none of these conditions did palmitic acid or hydroxybutyrate modulate ABCA1 gene expression (Fig. 3 A – D ). Likewise, and in agreement with data from Wang and Oram (19), we did not see any suppressive effect of fatty acids or acetoacetate on ABCA1 gene expression in macrophages that were stimulated with Br-cAMP (data not shown). We veri fi ed the stimulatory effects of 22(R)-HC and 9- cis- RA as well as the inhibitory effects of EPA and acetoacetate in a luciferase reporter gene assay, which was performed on a construct that contained 0.968 kb of the human ABCA1 promotor (Fig. 4). As expected from previous reports (12 – 14), 22R-HC and 9- cis- RA increased the ABCA1 promotor activity by a factor up to 30 (Figs. 4 A and B ). EPA and acetoacetate suppressed these stimula- tory effects dose dependently by up to 80% (EPA, Figs. 4 C and D ) and 50% (acetoacetate, Figs. 4 E and F ), respectively. Using this reporter gene assay, we compared the suppressive effects of various fatty acid species at a concentration of 100 mol/l. Linoleic acid was most effec- tive ( Ϫ 80%), followed by arachidonic acid ( Ϫ 75%), EPA ( Ϫ 70%), and oleic acid ( Ϫ 40%). Palmitic acid had a modest but signi fi cant suppressive effect ( Ϫ 10%), whereas stearic acid exerted a moderate but also signi fi cant stimulatory effect ( ϩ 10%, Fig. 5). Our observations raised the question as to whether unsaturated fatty acids and acetoacetate regulate the expression of ABCA1 indirectly via regulation of LXR ␣ or RXR ␣ gene expression. Such a mechanism was previously demonstrated to be responsible for the upregulation of ABCA1 by agonists of peroxisome proliferator – activated receptor (PPAR)- ␣ (i.e., fi brates) and PPAR ␥ (i.e., glita- zones) (20). Neither oleic acid nor EPA nor acetoacetate regulated the expression of LXR ␣ or RXR ␣ (data not shown). Immunoprecipitation and subsequent Western blot analysis clearly detected ABCA1 in RAW264.7 cells (Fig. 6 A ), which were cultivated in the presence of an oxysterol. However, the cellular ABCA1 protein concentration was severely decreased when these cells were incubated in the additional presence of free fatty acids or acetoacetate (Fig. 6 A ). The functional relevance of ABCA1 downregulation by free fatty acids and ketone bodies was investigated in cholesterol ef fl ux experiments (Fig. 6 B ). Lipid- free apoA-I induced signi fi cant cholesterol ef fl ux from RAW264.7 cells. As reported previously, cholesterol ef fl ux to apoA-I was stimulated by oxysterols (11 – 13). The stimulatory effect of oxysterols was completely inhibited by 500 mol/l oleic acid, 100 mol/l EPA, and 10 mmol/l acetoacetate (Fig. 6 B ). Interestingly, the expression of another important gene involved in cholesterol ef fl ux, scavenger receptor BI, was not affected by either fatty acids or acetoacetate (data not shown). Here we have demonstrated that ABCA1 is tremendously downregulated in an animal model of diabetes (Fig. 1). As the most likely explanation, we found suppressive effects of PUFAs, oleic acid, and acetoacetate on ABCA1 gene expression in vitro (Figs. 2 – 5). Interestingly, the saturated palmitic and stearic acids as well as the ketone body 3-hydroxybutyrate did not alter ABCA1 gene expression so that the inhibitory effects appear to be speci fi c for unsaturated fatty acids and acetoacetate. Suppression of ABCA1 gene expression by unsaturated fatty acids became most obvious in the additional presence of an oxysterol (Fig. 3) but did not occur in the presence of Br-cAMP (data not shown, 19). This raised the possibility that unsaturated fatty acids downregulate ABCA1 via suppression or inhibition of the nuclear transcription factor LXR ␣ , which is activated by oxysterols and which upregulates ABCA1 (12,13,21). Free fatty acids were previously shown to interact with LXR ␣ on both the transcriptional and the posttranslational level (22,23). Free fatty acids induce the transcription of the LXR gene via activation of PPARs (22,24). However, unsaturated fatty acids do not suppress ABCA1 via transcriptional regulation of LXR ␣ , since in our experiments oleic acid and EPA did not modulate the expression of LXR ␣ (data not shown) and since PPAR ␣ , PPAR ␥ , and PPAR ␦ rather upregulate ABCA1 via stimulation of LXR ␣ transcription (14,25). On the posttranslational level, unsaturated fatty acids antag- onize the activation of LXR ␣ by oxysterols and thereby inhibit the transcription of the sterol regulatory element- binding protein-1c (23,26). This posttranslational inhibition of LXR ␣ well explains the transcriptional suppression of ABCA1 by unsaturated fatty acids. Interestingly, PUFAs turned out as more suppressive than the monounsaturated oleic acid, whereas saturated fatty acids exerted only moderate or even no suppressive effects at all. This order is in agreement with the effects of dietary fatty acids on HDL cholesterol. Compared with habitual Western diets being rich in saturated fatty acids, diets rich in PUFAs substantially lower HDL cholesterol, whereas diets rich in oleic acid have only a ...
Context 4
... We incubated HepG2 hepatocytes (Fig. 2 A ) or RAW264.7 macrophages (Fig. 2 B ) for 18 h with increasing dosages of insulin, glucose, fatty acids, or ketone bodies. Addition of insulin or glucose into the cell culture medium had no consistent effects on the expression of ABCA1 in either hepatocytes or macrophages. By contrast, the polyunsaturated EPA as well as the ketone body acetoacetate dose-dependently decreased the expression of ABCA1 in both cell lines (Figs. 2 A and B ). The suppressive effects of these fatty acids became most evident in those cells where ABCA1 expression was up- regulated with an oxysterol, i.e., an activator of the nuclear transcription factor LXR ␣ , which was previously shown to induce the ABCA1 gene (12,13). In HepG2 cells, the stimulatory effect of 22R-HC (12,13) on ABCA1 was inhibited by ϳ 80% ( P Ͻ 0.01), 60% ( P Ͻ 0.01), and 40% ( P Ͻ 0.05) in the additional presence of physiological dosages of EPA (100 mol/l), oleic acid (500 mol/l), or acetotac- etate (10 mmol/l), respectively (Fig. 3 A ). In RAW264.7 cells, the respective numbers were 95, 80, and 60% (all P Ͻ 0.01; Fig. 3 B ). Also, in the presence of 9- cis- RA, i.e., an activator of the nuclear transcription factor RXR ␣ that forms heterodimers with LXR ␣ and thereby also induces the ABCA1 gene (14), EPA, oleic acid, and acetoacetate suppressed ABCA1 gene expression in HepG2 and RAW by 50 – 60% (Figs. 3 C and D , all signi fi cant at a level of P Ͻ 0.05 or P Ͻ 0.01, except acetoacetate in HepG2 cells). In none of these conditions did palmitic acid or hydroxybutyrate modulate ABCA1 gene expression (Fig. 3 A – D ). Likewise, and in agreement with data from Wang and Oram (19), we did not see any suppressive effect of fatty acids or acetoacetate on ABCA1 gene expression in macrophages that were stimulated with Br-cAMP (data not shown). We veri fi ed the stimulatory effects of 22(R)-HC and 9- cis- RA as well as the inhibitory effects of EPA and acetoacetate in a luciferase reporter gene assay, which was performed on a construct that contained 0.968 kb of the human ABCA1 promotor (Fig. 4). As expected from previous reports (12 – 14), 22R-HC and 9- cis- RA increased the ABCA1 promotor activity by a factor up to 30 (Figs. 4 A and B ). EPA and acetoacetate suppressed these stimula- tory effects dose dependently by up to 80% (EPA, Figs. 4 C and D ) and 50% (acetoacetate, Figs. 4 E and F ), respectively. Using this reporter gene assay, we compared the suppressive effects of various fatty acid species at a concentration of 100 mol/l. Linoleic acid was most effec- tive ( Ϫ 80%), followed by arachidonic acid ( Ϫ 75%), EPA ( Ϫ 70%), and oleic acid ( Ϫ 40%). Palmitic acid had a modest but signi fi cant suppressive effect ( Ϫ 10%), whereas stearic acid exerted a moderate but also signi fi cant stimulatory effect ( ϩ 10%, Fig. 5). Our observations raised the question as to whether unsaturated fatty acids and acetoacetate regulate the expression of ABCA1 indirectly via regulation of LXR ␣ or RXR ␣ gene expression. Such a mechanism was previously demonstrated to be responsible for the upregulation of ABCA1 by agonists of peroxisome proliferator – activated receptor (PPAR)- ␣ (i.e., fi brates) and PPAR ␥ (i.e., glita- zones) (20). Neither oleic acid nor EPA nor acetoacetate regulated the expression of LXR ␣ or RXR ␣ (data not shown). Immunoprecipitation and subsequent Western blot analysis clearly detected ABCA1 in RAW264.7 cells (Fig. 6 A ), which were cultivated in the presence of an oxysterol. However, the cellular ABCA1 protein concentration was severely decreased when these cells were incubated in the additional presence of free fatty acids or acetoacetate (Fig. 6 A ). The functional relevance of ABCA1 downregulation by free fatty acids and ketone bodies was investigated in cholesterol ef fl ux experiments (Fig. 6 B ). Lipid- free apoA-I induced signi fi cant cholesterol ef fl ux from RAW264.7 cells. As reported previously, cholesterol ef fl ux to apoA-I was stimulated by oxysterols (11 – 13). The stimulatory effect of oxysterols was completely inhibited by 500 mol/l oleic acid, 100 mol/l EPA, and 10 mmol/l acetoacetate (Fig. 6 B ). Interestingly, the expression of another important gene involved in cholesterol ef fl ux, scavenger receptor BI, was not affected by either fatty acids or acetoacetate (data not shown). Here we have demonstrated that ABCA1 is tremendously downregulated in an animal model of diabetes (Fig. 1). As the most likely explanation, we found suppressive effects of PUFAs, oleic acid, and acetoacetate on ABCA1 gene expression in vitro (Figs. 2 – 5). Interestingly, the saturated palmitic and stearic acids as well as the ketone body 3-hydroxybutyrate did not alter ABCA1 gene expression so that the inhibitory effects appear to be speci fi c for unsaturated fatty acids and acetoacetate. Suppression of ABCA1 gene expression by unsaturated fatty acids became most obvious in the additional presence of an oxysterol (Fig. 3) but did not occur in the presence of Br-cAMP (data not shown, 19). This raised the possibility that unsaturated fatty acids downregulate ABCA1 via suppression or inhibition of the nuclear transcription factor LXR ␣ , which is activated by oxysterols and which upregulates ABCA1 (12,13,21). Free fatty acids were previously shown to interact with LXR ␣ on both the transcriptional and the posttranslational level (22,23). Free fatty acids induce the transcription of the LXR gene via activation of PPARs (22,24). However, unsaturated fatty acids do not suppress ABCA1 via transcriptional regulation of LXR ␣ , since in our experiments oleic acid and EPA did not modulate the expression of LXR ␣ (data not shown) and since PPAR ␣ , PPAR ␥ , and PPAR ␦ rather upregulate ABCA1 via stimulation of LXR ␣ transcription (14,25). On the posttranslational level, unsaturated fatty acids antag- onize the activation of LXR ␣ by oxysterols and thereby inhibit the transcription of the sterol regulatory element- binding protein-1c (23,26). This posttranslational inhibition of LXR ␣ well explains the transcriptional suppression of ABCA1 by unsaturated fatty acids. Interestingly, PUFAs turned out as more suppressive than the monounsaturated oleic acid, whereas saturated fatty acids exerted only moderate or even no suppressive effects at all. This order is in agreement with the effects of dietary fatty acids on HDL cholesterol. Compared with habitual Western diets being rich in saturated fatty acids, diets rich in PUFAs substantially lower HDL cholesterol, whereas diets rich in oleic acid have only a little HDL cholesterol – decreasing effect (27). The suppressive effect of acetoacetate on ABCA1 gene expression was more obvious in macrophages than in hepatocytes (Figs. 3 – 5). Like PUFAs and oleic acid, acetoacetate did not suppress the expression of LXR ␣ or RXR ␣ . Downregulation of ABCA1 by acetoacetate is, to the best of our knowledge, the fi rst example of a gene regulatory effect exerted by this ketone body. Previous in vivo and in vitro studies demonstrated that ketoacidosis decreases hepatic apoA-I gene expression in rats (28). However, this effect was linked to low pH rather than to ketone bodies (28). In vitro we also observed that acidosis suppresses ABCA1 gene expression (data not shown); however, in our experiments the addition of acetoacetate did not acidify the cell culture medium. Moreover, in those experiments, suppression of apoA-I was accomplished by butyrate (28), which did not suppress ABCA1 in our experiments. Thus, the suppressive effect appears to be speci fi c for acetoacetate. Our data do not allow any conclusion of whether acetoacetate exerts the inhibitory effect directly by itself or indirectly by a metabolite or a signal elicited thereof. It is unlikely that the regulatory effect of acetoacetate is exerted by a metabolite because ketolysis is a hallmark of heart and skeletal muscle as well as kidney and brain, which in contrast to liver and macrophages have a high activity of the rate-limiting enzyme in ketolysis, i.e., succinyl-CoA-oxoacid transferase (29,30). Because acetoacetate but not  -hydroxybutyrate downregulates the expression of ABCA1 and because the suppressive effect of acetoacetate was more obvious in macrophages than in hepatocytes, it is interesting to note that acetoacetate by contrast to  -hydroxybutyrate generates oxygen radicals in cells (31). Because oxygen radicals are known to regulate gene expression via various signal- ing pathways (32), it will be important to investigate the effects of oxygen radicals on the expression of ABCA1. Our fi ndings contribute to the understanding of the pathogenesis of the low HDL cholesterol syndrome in patients with latent or manifest type 2 diabetes. Insulin was previously shown to stimulate hepatic apoA-I gene expression (33,34), to inhibit hepatic VLDL production (35,36), and to stimulate the release of lipoprotein lipase from adipose tissue (37). Insulin resistance hence reduces the hepatic production of nascent HDL and induces hypertriglyceridemia, which secondarily causes low HDL cholesterol levels via disturbed release of surface remnants (1,2,38) and by enhancing the exchange of triglycerides from VLDL against cholesteryl esters from HDL (1,2,39). Free fatty acids were previously shown to downregulate the expression of apoA-I (40) and to upregulate the expression of cholesteryl ester transfer protein (41). In addition, we have demonstrated here that unsaturated fatty acids and acetoacetate suppress the expression of ABCA1 in liver and in macrophages. By the latter effect, hyperacylemia and ketosis in diabetes and insulin resistance will probably not only interfere with the formation of HDL but also facilitate lipid accumulation in vascular macrophages. Patients with type 1 diabetes have increased cardiovascular risk but normal or even slightly elevated levels of HDL cholesterol (42). This is, at fi rst sight, in partial contrast ...
Citations
... The cellular CEC was measured as described previously (30)(31)(32)(33)(34). Briefly, J774A.1 cells were radiolabeled with 5 uCi/mL of [1,2-3H] cholesterol (PerkinElmer, Waltham, MA, United States) for 24 h. ...
Policosanol supplementation has been reported to increase high-density lipoprotein (HDL)-cholesterol (HDL-C). However, the association between Cuban policosanol supplementation and HDL cholesterol efflux capacity (CEC), an important function of HDL, remains unclear. We performed a lipoprotein analysis investigating 32 Japanese healthy participants (placebo, n = 17 or policosanol supplementation for 12 weeks, n = 15) from a randomized Cuban policosanol clinical trial. First, HDL CEC and HDL-related factors were measured before and after policosanol supplementation. Then, through electron microscopy after ultracentrifugation and high-performance liquid chromatography, HDL morphology and subclass were analyzed, respectively. Finally, the effects of policosanol supplementation regarding HDL function, HDL-related factors, and HDL morphology/component were examined. Cuban policosanol considerably increased the HDL CEC and HDL-C and apolipoprotein A-I (ApoA-I) levels. Furthermore, policosanol supplementation led to larger HDL particles, increased cholesterol content in larger HDL particles, and reduced triglyceride content in smaller HDL particles. In participants with high baseline HDL-C levels, the policosanol effects for HDL CEC are observed. HDL CEC fluctuation induced by policosanol was highly associated with HDL-C and ApoA-I changes. In conclusion, for the first time, we demonstrated that policosanol supplementation increased the HDL CEC in healthy participants.
... THP-1 cells treated with PMA were radiolabeled with [1,2-3 H] cholesterol (Perki-nElmer, Waltham, MA, USA). The cellular cholesterol efflux was measured as described previously [31,34]. ...
... cAMP and ligands of the nuclear transcription factors LXRα and RXRα have been identified as enhancers of ABCA1 gene expression, and cAMP has been shown to activate apoA1-mediated cellular cholesterol efflux [7,31,34,48]. It is well known that GLP-1R is a G-protein-coupled receptor, and its activation increases the intracellular cAMP levels via the activation of adenylate cyclase [49]. ...
Cholesterol efflux is a major atheroprotective function of high-density lipoproteins (HDLs) which removes cholesterol from the foam cells of lipid-rich plaques in Type 2 diabetes. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin phosphate increases plasma glucagon-like peptide-1 (GLP-1) concentrations and is used to treat Type 2 diabetes. GLP-1 plays an important role in regulating insulin secretion and expression via the GLP-1 receptor (GLP-1R), which is expressed in pancreatic islets as well as freshly isolated human monocytes and THP-1 cells. Here, we identified a direct role of GLP-1 and DPP-4 inhibition in HDL function. Cholesterol efflux was measured in cultivated phorbol 12-myristate 13-acetate-treated THP-1 cells radiolabeled with 3H-cholesterol and stimulated with liver X receptor/retinoid X receptor agonists. Contrary to vildagliptin, sitagliptin phosphate together with GLP-1 significantly (p < 0.01) elevated apolipoprotein (apo)A1-mediated cholesterol efflux in a dose-dependent manner. The sitagliptin-induced increase in cholesterol efflux did not occur in the absence of GLP-1. In contrast, adenosine triphosphate-binding cassette transporter A1 (ABCA1) mRNA and protein expressions in the whole cell fraction were not changed by sitagliptin in the presence of GLP-1, although sitagliptin treatment significantly increased ABCA1 protein expression in the membrane fraction. Furthermore, the sitagliptin-induced, elevated efflux in the presence of GLP-1 was significantly decreased by a GLP-1R antagonist, an effect that was not observed with a protein kinase A inhibitor. To our knowledge, the present study reports for the first time that sitagliptin elevates cholesterol efflux in cultivated macrophages and may exert anti-atherosclerotic actions that are independent of improvements in glucose metabolism. Our results suggest that sitagliptin enhances HDL function by inducing a de novo HDL synthesis via cholesterol efflux.
... A decrease in the expression and functional activity of these transporters is associated with the formation of "foam cells" and the progression of atherosclerosis. Unsaturated fatty acids such as palmitoleic, oleic, and linoleic acids have been shown to reduce ABCA1 expression at transcriptional as well as posttranscriptional levels, reducing cholesterol efflux (134,135). Eicosapentaenoic and oleic acids also reduced ABCA1 promoter activity in macrophages. Unsaturated fatty acids also suppressed ABCG1 gene expression through a mechanism that involves LXR/RXR binding to promoters (136). ...
Cardiovascular diseases are one of the most important problems of modern medicine. They are associated with a large number of health care visits, hospitalizations and mortality. Prevention of atherosclerosis is one of the most effective strategies and should start as early as possible. Correction of lipid metabolism disorders is associated with definite clinical successes, both in primary prevention and in the prevention of complications of many cardiovascular diseases. A growing body of evidence suggests a multifaceted role for polyunsaturated fatty acids. They demonstrate a variety of functions in inflammation, both participating directly in a number of cellular processes and acting as a precursor for subsequent biosynthesis of lipid mediators. Extensive clinical data also support the importance of polyunsaturated fatty acids, but all questions have not been answered to date, indicating the need for further research.
... Lipid metabolites other than sterols also can modulate ABCA1 expression by the nuclear receptor system. Polyunsaturated fatty acids decreased the expression of ABCA1 in RAW264.7 macrophages [147] by acting as antagonists to oxysterol binding to LXRα [148] and by modulating the histone acetylation state [149]. Geranylgeranyl pyrophosphate (GGPP), an endogenous intermediate of the mevalonate pathway that isoprenylates proteins, has been shown to act as a strong inhibitor of ABCA1 mRNA synthesis in THP-1 cells and Caco-2 intestinal cells [150]. ...
ATP-binding cassette subfamily A member 1 (ABCA1) protein plays an essential role in a variety of events, such as cholesterol and phospholipid efflux, nascent high-density lipoprotein (HDL) biosynthesis, phospholipid translocation. Thus, there has been much research activity aimed at understanding the molecular mechanisms of regulating ABCA1 expression. In this review, we first discuss ABCA1 structure, tissue distribution, cellular localization and trafficking, as well as its function. Furthermore, current understanding of the molecular mechanisms involved in the regulation of ABCA1 expression are summarized. ABCA1 transcriptional regulation is mediated by a very complicated system, including nuclear receptor systems, factors binding to other sites in the ABCA1 promoter, cytokines, hormones, growth factors, lipid metabolites, enzymes, and other messengers/factors/pathways. In addition, ABCA1 post-transcriptional regulation is mediated by microRNA, long noncoding RNA, RNA-binding proteins, proteases, fatty acids, PDZ proteins, signaling proteins, and other factors. Compared to the transcriptional regulation of ABCA1 which is well established, the post-transcriptional regulation of ABCA1 expression is poorly understood.
... Nuclear receptors LXR and RXR, acting as a heterodimer, bind to the DR4 element in the ABCA1 promoter and activate its transcription [69][70][71][72][73]. LXR/RXR is activated by small hydrophobic ligands, such as retinoic acid and hydroxycholesterol, inducing ABCA1 expression, cholesterol efflux, and promoting RCT. At the same time, unsaturated fatty acids suppress the stimulatory effects of oxysterols and retinoids on the expression of ABCA1 mRNA, apparently also through the DR4 element [74,75]. Interestingly, LXR/RXR also activates stearoyl-CoA desaturase, which can generate ABCA1-suppressing monounsaturated fatty acids from their saturated precursors. ...
Atheroprotective properties of human plasma high-density lipoproteins (HDLs) are determined by their involvement in reverse cholesterol transport (RCT) from the macrophage to the liver. ABCA1, ABCG1, and SR-BI cholesterol transporters are involved in cholesterol efflux from macrophages to lipid-free ApoA-I and HDL as a first RCT step. Molecular determinants of RCT efficiency that may possess diagnostic and therapeutic meaning remain largely unknown. This review summarizes the progress in studying the genomic variants of ABCA1, ABCG1, and SCARB1, and the regulation of their function at transcriptional and post-transcriptional levels in atherosclerosis. Defects in the structure and function of ABCA1, ABCG1, and SR-BI are caused by changes in the gene sequence, such as single nucleotide polymorphism or various mutations. In the transcription initiation of transporter genes, in addition to transcription factors, long noncoding RNA (lncRNA), transcription activators, and repressors are also involved. Furthermore, transcription is substantially influenced by the methylation of gene promoter regions. Post-transcriptional regulation involves microRNAs and lncRNAs, including circular RNAs. The potential biomarkers and targets for atheroprotection, based on molecular mechanisms of expression regulation for three transporter genes, are also discussed in this review.
... Tsujita et al. (2017) concluded that ABCA1 protein expression was upregulated in the liver of diabetic animals without an apparent increase of its mRNA. Most others studies have found the mRNA and protein levels of ABCA1 decreased in the liver of diabetic mice, as well as in HepG2 or RAW264 cells exposed to HG (Tu and Albers, 2001;Uehara et al., 2002), a conclusion that is consistent with our results. However, no rational interpretation has been proposed for these discrepancies. ...
Background
The underlying mechanisms by which diabetes and dyslipidemia contribute to diabetic nephropathy (DN) are not fully understood. In this study, we aimed to investigate the role of high glucose (HG) on intracellular cholesterol accumulation in glomerular endothelial cells (GEnCs) and its potential mechanism.
Methods
Oil red O staining, RT-qPCR, Western blotting, and immunocytofluorescence analyses were used to determine cholesterol accumulation and the expressions of LXRs and ABCA1 in GEnCs under high cholesterol (HC) and/or HG conditions, and the effect of these treatments was compared to that of low glucose without adding cholesterol. LncRNA microarrays were used to identify a long non-coding RNA (LncRNA OR13C9), of which levels increased in cells treated with the LXR agonist, GW3965. Fluorescence in situ hybridization (FISH) was conducted to confirm subcellular localization of LncOR13C9 and a bioinformatics analysis was used to identify competing endogenous RNA (ceRNA) regulatory networks between LncOR13C9 and microRNA-23a-5p (miR-23a-5p). Gain and loss of function, rescue assay approaches, and dual-luciferase reporter assay were conducted to study interactions between LncOR13C9, miR-23a-5p, and ABCA1.
Results
We showed that HG could decrease the response ability of GEnCs to cholesterol load, specifically that HG could downregulate LXRs expression in GEnCs under cholesterol load and that the decrease in LXRs expression suppressed ABCA1 expression and increased cholesterol accumulation. We focused on the targets of LXRs and identified a long non-coding RNA (LncOR13C9) that was downregulated in GEnCs grown in HG and HC conditions, compared with that grown in HC conditions. We speculated that LncRNAOR13C9 was important for LXRs to increase cholesterol efflux via ABCA1 under HC. Furthermore, using gain of function, loss of function, and rescue assay approaches, we showed that LncOR13C9 could regulate ABCA1 by inhibiting the action of miR-23a-5p in the LXR pathway. Furthermore, dual-luciferase reporter assay was conducted to study the interaction of LncOR13C9 with miR-23a-5p.
Conclusion
Overall, our study identified the LXRs/LncOR13C9/miR23A-5p/ABCA1 regulatory network in GEnCs, which may be helpful to better understand the effect of HG on cholesterol accumulation in GEnCs under cholesterol load and to explore new therapeutic tools for the management of DN patients.
... However, this lack of association between HDL-C (and its subfractions) and CAC after triglycerides adjustment shows a different scenario regarding subclinical atherosclerosis. It is known there is a direct LDL and triglycerides interplay in HDL metabolism, through the continuous cholesterol and TG exchange with apo-B lipoprotein particles by the activate CETP, and the inhibition of expression (44) and proteasomal degradation (45) of adenosine triphosphate-binding cassette transporter A1 (ABCA1) by unsaturated free fatty acids in the liver, leading to less HDL generation. Therefore, the strong attenuation of the association of HDL-c (and also of its subfractions) with coronary calcification might be explained by this close interaction in the metabolic pathway. ...
Background:
Although elevated high-density lipoprotein cholesterol (HDL-C) is considered protective against atherosclerotic cardiovascular disease, no causal relationship has been demonstrated. HDL-C comprises a group of different subfractions that might have different effects on atherosclerosis. Our objective was to investigate the association between HDL-C subfractions with the coronary artery calcium (CAC) score.
Methods:
We included 3,674 (49.8 ± 8.3 years, 54% women) participants from the ELSA-Brasil study who had no prior history of CVD and were not currently using lipid-lowering medications. We measured the fasting lipoprotein cholesterol fractions (in mmol/l) by a zonal ultracentrifugation method (VAP). We analyzed the independent predictive values of total HDL-C, HDL2-C, and HDL3-C subfractions and in the HDL2-C/HDL3-C ratio using linear regression to predict Ln(CAC+1) and logistic regression to predict the presence of CAC.
Results:
Overall 912 (24.8%) of the participants had CAC>0, and 294 (7.7%) had CAC>100. The mean total HDL-C, HDL2-C, and HDL3-C were: 1.42 ± 0.37, 0.38 ± 0.17 and 1.03 ± 0.21 mmol/l, respectively. Individuals with CAC>0 had lower levels of total HDL-C as well as of each subfraction (p < 0.001). When adjusted for age, gender, smoking, hypertension, alcohol use, physical activity, and LDL-C, we observed an inverse association between HDL-C and its subfractions and CAC (p < 0.05). However, by adding triglycerides in the adjustment, neither total HDL-C nor its subfractions remained independently associated with the presence or extent of CAC.
Conclusion:
In this cross-sectional analysis, neither the total HDL-C nor its subfractions (HDL2-C and HDL3-C, as well as HDL2-C/HDL3-C ratio) measured by VAP are independently associated with the presence or extent of coronary calcification.
... This is in line with our observation in diabetic subjects with European background. On the other hand, ketone bodies such as acetoacetate may also inhibit insulin secretion by suppressing expression of ABCA1 (a key player in cellular cholesterol efflux) and promote accumulation of intracellular cholesterol 42,43 . We speculate that our observation of positive associations between HbA1c and circulating acetoacetate in African participants may be linked to the insufficient ABCA1-mediated cholesterol efflux capacity. ...
Background
The prevalence of type 2 diabetes mellitus (T2DM) varies significantly across ethnic groups. A better understanding of the mechanisms underlying the variation in different ethnic groups may help to elucidate the pathophysiology of T2DM. The present work aims to generate a hypothesis regarding “why do subjects with African background have excess burden of T2DM?”.
Methods
In the current study, we performed metabolite profiling of plasma samples derived from 773 subjects of three ethnic groups (Dutch with European, Ghanaian and African Surinamese background). We performed Bayesian lognormal regression analyses to assess associations between HbA1c and circulating metabolites.
Results
Here we show that subjects with African Surinamese and Ghanaian background had similar associations of HbA1c with circulating amino acids and triglyceride-rich lipoproteins as subjects with European background. In contrast, subjects with Ghanaian and African Surinamese background had different associations of HbA1c with acetoacetate, small LDL particle and small HDL particle concentrations, compared to the subjects with European background.
Conclusions
On the basis of the observations, we hypothesize that the excess burden of T2DM in subjects with African background may be due to impaired cholesterol efflux capacity or abnormal cholesterol uptake.
... Significantly elevated unsaturated fatty acids in patients with diabetes mellitus have been shown to phosphorylate ABCA1 through phospholipase D2 pathway and reduce its stability, thus affecting its function [27] and reducing cholesterol reverse transport. The increased SAA in T2DM patients binds to HDL and increases its affinity to macrophages by 3 to 4 times, while the affinity to hepatocytes decreases. ...
... UFAs and cholesterol efflux [56][57][58][59]. Dose-dependent effects of UFAs on inhibiting cholesterol efflux from mouse macrophages by downregulating ABCA1 mRNA [56] and/or increasing degradation of ABCA1 protein [57,59] have been observed in in vitro studies. ...
... UFAs and cholesterol efflux [56][57][58][59]. Dose-dependent effects of UFAs on inhibiting cholesterol efflux from mouse macrophages by downregulating ABCA1 mRNA [56] and/or increasing degradation of ABCA1 protein [57,59] have been observed in in vitro studies. Indeed, the overexpression of either Apo A1 or ABCA1 can result in an increase in cholesterol efflux and a decrease in fatty acid synthesis [60]. ...
Aortic stiffness during cardiac contraction is defined by the rigidity of the aorta and the elastic resistance to deformation. Recent studies suggest that aortic stiffness may be associated with changes in cholesterol efflux in endothelial cells. This alteration in cholesterol efflux may directly affect endothelial function, extracellular matrix composition, and vascular smooth muscle cell function and behavior. These pathological changes favor an aortic stiffness phenotype. Among all of the proteins participating in the cholesterol efflux process, ATP binding cassette transporter A1 (ABCA1) appears to be the main contributor to arterial stiffness changes in terms of structural and cellular function. ABCA1 is also associated with vascular inflammation mediators implicated in aortic stiffness. The goal of this mini review is to provide a conceptual hypothesis of the recent advancements in the understanding of ABCA1 in cholesterol efflux and its role and association in the development of aortic stiffness, with a particular emphasis on the potential mechanisms and pathways involved.
