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Role of Scavenger Receptor CD36 in Cholesterol Metabolism in Hepatocytes.
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CANADIAN JOURNAL OF DIABETES
POSTER PRESENTATIONS 195
196 Role of Scavenger Receptor CD36 in Cholesterol Metabolism in Hepatocytes. AMÉLIE RODRIGUEWAY* , ANNIE DEMERS, CAROLE GAROFALO, ÉMILE LÉVY and ANDRÉ TREMBLAY, Department of Biochemistry and CHU Ste-Justine, University of Montreal, Montréal, QC.
Molecular Mecanisms Involved in PPAR Activation by the Ghrelin Receptor in Macrophage. DEMERS ANNIE*1,2, AMÉLIE RODRIGUE-WAY1,3, HUY ONG2, ANDRÉ TREMBLAY1,3 1CHU Ste-Justine and 2Faculty of Pharmacy and 2Department of biochemistry, University of Montreal, Montréal, QC The peroxisome proliferator-activated receptor (PPAR) family of nuclear hormone receptors is involved in the regulation of key pathways linked to lipid and glucose metabolism, and energy homeostasis. Recently, we have reported a novel regulatory pathway involved in macrophage cholesterol metabolism by which the activation of the ghrelin receptor (GHS-R1a) and the scavenger receptor CD36 resulted in an enhanced transcriptional activation of PPAR, leading to antiatherogenic properties in a mouse model of atherosclerosis. Such activation of PPAR was independent of its hormone binding domain or activation function AF-2 but rather to an enhanced AF-1 activity of PPAR. In the present study, we investigated the implication of GHS-1a signaling on the homeostasis function of PPAR in macrophages. Using cell reporter assays, we showed that the activation of GHS-R1a by ghrelin activates PPAR. Neither the hormone binding domain, nor the serine 112, previously described to reduce PPAR sensitivity to its ligands, appears to be involved in PPAR activation by ghrelin. Consistent with this, activation of GHSR1a lead to an enhanced phosphorylation of PPAR which was preserved using the PPARS112A mutant in 293 cells, suggesting that the inhibitory Ser-112 site is not affecting PPAR response to ghrelin. Treatment of differentiated human THP-1 macrophages with ghrelin increases PPAR phosphorylation, indicating a role for protein kinases in targeting PPAR through GHS-R1a. We found that in response to ghrelin, Akt and Erk kinases were activated in THP-1 cells with a concomitant recruitment of the Src tyrosine kinase Fyn to the GHS-R1a receptor. Interestingly, the use of Src kinase inhibitor PP2 highlights an important role of Fyn kinase to affect the MAPK but not the Akt pathway in response to ghrelin, suggesting a role of Fyn to restrain the inhibitory effect of Erk on PPAR. Moreover, the interaction between PPAR and Akt was not modulated by PP2. These results suggest that Fyn kinase recruitment to GHS-R1a is important to support a concerted action of both Erk and Akt pathways in order to finely tune PPAR response to ghrelin in macrophages.
266 CSEM Metabolism
The identification of excess body weight as a major risk factor, the epidemic of obesity and diabetes and their increasing prevalence in children indicate that pathologies associated to metabolic syndrome will continue to impact human health. The scavenger receptor CD36 fulfills a variety of function depending on its tissue location. The uptake of oxidized lipoproteins by CD36 in macrophages has been described as the detrimental step in foam cell formation and atherogenesis, while in adipocytes it is mainly responsible for the intake of long-chain fatty acids. We have reported that activation of CD36 by the growthhormone secretagogue hexarelin, stimulates cholesterol efflux in a manner dependent on nuclear receptor PPARJ in macrophages. In addition, we have determined that such interaction with CD36 promotes mitochondrial biogenesis and a thermogenic phenotype in white adipocytes by increasing fatty acid oxidation. In the liver, little is known about the influence of CD36 on lipid metabolism. Here, we report that the interaction of hexarelin with CD36 resulted in a reduction of total cellular cholesterol in cultured HepG2 hepatocytes. Western analysis showed a decrease in the levels of the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase (HMGR). This decrease was explained by an enhanced ubiquitination of HMGR, a key step in its degradation. HepG2 cells treated with hexarelin showed an increase in the expression of several genes under the control of SREBP2 (sterol regulatory element binding protein 2), a transcription factor activated upon sterol depletion. Luciferase assays using the SREBP-binding element found in the HMGR promoter region showed an increase in the activity of SREBPs by hexarelin. In conclusion, the interaction of CD36 resulted in an accelerated degradation of HMGR and a decrease of cellular cholesterol. The rapid compensatory increase in the expression of SREBP2-target genes was not sufficient to restore the cellular cholesterol pool following treatment with hexarelin. Therefore, we propose a novel role for CD36 in the metabolism of cholesterol in hepatocytes.
197 Estrogen prevents beta-cell failure and diabetes in ZDF male rats: a role for glycerolipid/fatty acid cycling VIVIANE DELGHINGARO-AUGUSTO1, MARTIN G. LATOUR1, MARIE-LINE PEYOT1, JULIEN LAMONTAGNE1, ROXANE LUSSIER1, THIERRY ALQUIER1, FRANK MAUVAIS-JARVIS2, MARC PRENTKI1. 1 Molecular Nutrition Unit and the Montreal Diabetes Research Center, Departments of Nutrition and Biochemistry, University of Montreal and the Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada. 2 ,Division of Endocrinolgy, Metabolism and Molecular Medicine, Feinberg School of Medicine, Northwestern University, Chicago, USA. In most rodent models of diabetes, females are protected against betacell failure and hyperglycemia. In the male Zucker Fatty rats (ZDF), over secretion of insulin to compensate for peripheral insulin resistance is limited leading to progressive hyperglycemia and overt diabetes. Recently, we have shown that estrogen (E2) treatment prevents the development of diabetes in male ZDF rats. Here we investigate the mechanisms mediating the beneficial effects of E2 treatment on lipid and glucose metabolism in isolated islets from ZDF (sham or E2 treated) and Zucker lean (ZL) rats three weeks after treatment. At 9 weeks of age, sham-ZDF rats developed hyperglycaemia (~ 20 mmol/l) and hypoinsulinemia whereas ZDF-E2 remained normoglycemic (~ 6.5 mmol/l), but still hyperlipidemic. Furthermore, sham-ZDF rats had altered lipid partitioning with exaggerated triglyceride (TG) deposition when compared to ZL control rat islets. In addition, palmitate esterification into neutral glycerolipids was reduced in ZDF-E2 islets to similar levels as those observed in ZL controls. Basal glucose-stimulated lipolysis was increased in both ZDF islet groups when compared to ZL controls, whereas fatty acid oxidation was not changed. We propose that the beneficial effect of estradiol in the prevention of hyperglycemia in ZDF rats might be in part due to the normalization of lipid partitioning.
ABSTRACT #145
198
Hepatic Glucagon Receptor In Rats: Effect Of Type 1 Diabetes. JOHNATAN LAMANQUE*1, JESSICA MORISSETTE1, ALEXANDRE MELANÇON1, GENEVIÈVE ROBERT1, METABOLISM FRANÇOIS PÉRONNET2 AND CAROLE LAVOIE1. 1 Université du Québec à Trois-Rivières et 2Université de Montréal, Québec. The purpose of this study was to describe the density and binding properties of hepatic glucagon receptor in rats with insulin-treated type 1 diabetes (single injection of 80 mg/kg of streptozotocin i.p.). One week after the diabetes was established, and for the duration of the 8week experimentation period, blood glucose concentration was normalized by subcutaneous insulin 7-mm long implants. No difference was observed for weight (435 ± 15 and 423 ± 7 g) and blood glucose concentration (7.0 ± 1.2 and 7.7 ± 0.6 mmol/L) for the diabetes (n = 6) and control groups (n = 5) at the time of sacrifice. Plasma membranes were purified from liver, and saturation kinetics were obtained by incubation with (125)I-labelled glucagon. Saturating curves analysis indicated a similar glucagon receptor density in liver from diabetic and control animals (Bmax = 3.38 ± 0.48 and 3.09 ± 0.12 pmol/mg of proteins), but the affinity of the receptor was lower in diabetic rats (Kd = 1.02 ± 0.10 vs 0.33 ± 0.05 nM). These preliminary results suggest that in liver from type 1 diabetic rats treated with insulin, the density of hepatic glucagon receptor is normal but their affinity is lower than in liver from control animals. This finding could partly explain the lost of glucagon sensibility acquired with type 1 diabetes. Supported by Diabète Trois-Rivières, Diabète-Québec and NSERC.
CSEM
178 CSEM Other
ABSTRACT #78 CSEM METABOLISM
CANADIAN JOURNAL OF DIABETES
POSTER PRESENTATIONS 195
196 Role of Scavenger Receptor CD36 in Cholesterol Metabolism in Hepatocytes. AMÉLIE RODRIGUEWAY* , ANNIE DEMERS, CAROLE GAROFALO, ÉMILE LÉVY and ANDRÉ TREMBLAY, Department of Biochemistry and CHU Ste-Justine, University of Montreal, Montréal, QC.
Molecular Mecanisms Involved in PPAR Activation by the Ghrelin Receptor in Macrophage. DEMERS ANNIE*1,2, AMÉLIE RODRIGUE-WAY1,3, HUY ONG2, ANDRÉ TREMBLAY1,3 1CHU Ste-Justine and 2Faculty of Pharmacy and 2Department of biochemistry, University of Montreal, Montréal, QC The peroxisome proliferator-activated receptor (PPAR) family of nuclear hormone receptors is involved in the regulation of key pathways linked to lipid and glucose metabolism, and energy homeostasis. Recently, we have reported a novel regulatory pathway involved in macrophage cholesterol metabolism by which the activation of the ghrelin receptor (GHS-R1a) and the scavenger receptor CD36 resulted in an enhanced transcriptional activation of PPAR, leading to antiatherogenic properties in a mouse model of atherosclerosis. Such activation of PPAR was independent of its hormone binding domain or activation function AF-2 but rather to an enhanced AF-1 activity of PPAR. In the present study, we investigated the implication of GHS-1a signaling on the homeostasis function of PPAR in macrophages. Using cell reporter assays, we showed that the activation of GHS-R1a by ghrelin activates PPAR. Neither the hormone binding domain, nor the serine 112, previously described to reduce PPAR sensitivity to its ligands, appears to be involved in PPAR activation by ghrelin. Consistent with this, activation of GHSR1a lead to an enhanced phosphorylation of PPAR which was preserved using the PPARS112A mutant in 293 cells, suggesting that the inhibitory Ser-112 site is not affecting PPAR response to ghrelin. Treatment of differentiated human THP-1 macrophages with ghrelin increases PPAR phosphorylation, indicating a role for protein kinases in targeting PPAR through GHS-R1a. We found that in response to ghrelin, Akt and Erk kinases were activated in THP-1 cells with a concomitant recruitment of the Src tyrosine kinase Fyn to the GHS-R1a receptor. Interestingly, the use of Src kinase inhibitor PP2 highlights an important role of Fyn kinase to affect the MAPK but not the Akt pathway in response to ghrelin, suggesting a role of Fyn to restrain the inhibitory effect of Erk on PPAR. Moreover, the interaction between PPAR and Akt was not modulated by PP2. These results suggest that Fyn kinase recruitment to GHS-R1a is important to support a concerted action of both Erk and Akt pathways in order to finely tune PPAR response to ghrelin in macrophages.
266 CSEM Metabolism
The identification of excess body weight as a major risk factor, the epidemic of obesity and diabetes and their increasing prevalence in children indicate that pathologies associated to metabolic syndrome will continue to impact human health. The scavenger receptor CD36 fulfills a variety of function depending on its tissue location. The uptake of oxidized lipoproteins by CD36 in macrophages has been described as the detrimental step in foam cell formation and atherogenesis, while in adipocytes it is mainly responsible for the intake of long-chain fatty acids. We have reported that activation of CD36 by the growthhormone secretagogue hexarelin, stimulates cholesterol efflux in a manner dependent on nuclear receptor PPARJ in macrophages. In addition, we have determined that such interaction with CD36 promotes mitochondrial biogenesis and a thermogenic phenotype in white adipocytes by increasing fatty acid oxidation. In the liver, little is known about the influence of CD36 on lipid metabolism. Here, we report that the interaction of hexarelin with CD36 resulted in a reduction of total cellular cholesterol in cultured HepG2 hepatocytes. Western analysis showed a decrease in the levels of the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase (HMGR). This decrease was explained by an enhanced ubiquitination of HMGR, a key step in its degradation. HepG2 cells treated with hexarelin showed an increase in the expression of several genes under the control of SREBP2 (sterol regulatory element binding protein 2), a transcription factor activated upon sterol depletion. Luciferase assays using the SREBP-binding element found in the HMGR promoter region showed an increase in the activity of SREBPs by hexarelin. In conclusion, the interaction of CD36 resulted in an accelerated degradation of HMGR and a decrease of cellular cholesterol. The rapid compensatory increase in the expression of SREBP2-target genes was not sufficient to restore the cellular cholesterol pool following treatment with hexarelin. Therefore, we propose a novel role for CD36 in the metabolism of cholesterol in hepatocytes.
197 Estrogen prevents beta-cell failure and diabetes in ZDF male rats: a role for glycerolipid/fatty acid cycling VIVIANE DELGHINGARO-AUGUSTO1, MARTIN G. LATOUR1, MARIE-LINE PEYOT1, JULIEN LAMONTAGNE1, ROXANE LUSSIER1, THIERRY ALQUIER1, FRANK MAUVAIS-JARVIS2, MARC PRENTKI1. 1 Molecular Nutrition Unit and the Montreal Diabetes Research Center, Departments of Nutrition and Biochemistry, University of Montreal and the Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada. 2 ,Division of Endocrinolgy, Metabolism and Molecular Medicine, Feinberg School of Medicine, Northwestern University, Chicago, USA. In most rodent models of diabetes, females are protected against betacell failure and hyperglycemia. In the male Zucker Fatty rats (ZDF), over secretion of insulin to compensate for peripheral insulin resistance is limited leading to progressive hyperglycemia and overt diabetes. Recently, we have shown that estrogen (E2) treatment prevents the development of diabetes in male ZDF rats. Here we investigate the mechanisms mediating the beneficial effects of E2 treatment on lipid and glucose metabolism in isolated islets from ZDF (sham or E2 treated) and Zucker lean (ZL) rats three weeks after treatment. At 9 weeks of age, sham-ZDF rats developed hyperglycaemia (~ 20 mmol/l) and hypoinsulinemia whereas ZDF-E2 remained normoglycemic (~ 6.5 mmol/l), but still hyperlipidemic. Furthermore, sham-ZDF rats had altered lipid partitioning with exaggerated triglyceride (TG) deposition when compared to ZL control rat islets. In addition, palmitate esterification into neutral glycerolipids was reduced in ZDF-E2 islets to similar levels as those observed in ZL controls. Basal glucose-stimulated lipolysis was increased in both ZDF islet groups when compared to ZL controls, whereas fatty acid oxidation was not changed. We propose that the beneficial effect of estradiol in the prevention of hyperglycemia in ZDF rats might be in part due to the normalization of lipid partitioning.
ABSTRACT #145
198
Hepatic Glucagon Receptor In Rats: Effect Of Type 1 Diabetes. JOHNATAN LAMANQUE*1, JESSICA MORISSETTE1, ALEXANDRE MELANÇON1, GENEVIÈVE ROBERT1, METABOLISM FRANÇOIS PÉRONNET2 AND CAROLE LAVOIE1. 1 Université du Québec à Trois-Rivières et 2Université de Montréal, Québec. The purpose of this study was to describe the density and binding properties of hepatic glucagon receptor in rats with insulin-treated type 1 diabetes (single injection of 80 mg/kg of streptozotocin i.p.). One week after the diabetes was established, and for the duration of the 8week experimentation period, blood glucose concentration was normalized by subcutaneous insulin 7-mm long implants. No difference was observed for weight (435 ± 15 and 423 ± 7 g) and blood glucose concentration (7.0 ± 1.2 and 7.7 ± 0.6 mmol/L) for the diabetes (n = 6) and control groups (n = 5) at the time of sacrifice. Plasma membranes were purified from liver, and saturation kinetics were obtained by incubation with (125)I-labelled glucagon. Saturating curves analysis indicated a similar glucagon receptor density in liver from diabetic and control animals (Bmax = 3.38 ± 0.48 and 3.09 ± 0.12 pmol/mg of proteins), but the affinity of the receptor was lower in diabetic rats (Kd = 1.02 ± 0.10 vs 0.33 ± 0.05 nM). These preliminary results suggest that in liver from type 1 diabetic rats treated with insulin, the density of hepatic glucagon receptor is normal but their affinity is lower than in liver from control animals. This finding could partly explain the lost of glucagon sensibility acquired with type 1 diabetes. Supported by Diabète Trois-Rivières, Diabète-Québec and NSERC.
CSEM
178 CSEM Other
ABSTRACT #78 CSEM METABOLISM