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Involvement of heat shock factor-1(HSF1) in upregulation of plasminogen activator lnhihitor-1 in streptozotocin-induced diabetic, apolipoprotein E-knockout mice and fibroblasts from HSF1- knockout mice.
MONTREAL 2008 ABSTRACTS
ORAL PRESENTATIONS
29
I
30
SEBASTIE N
Increase in reactive oxygen species production, in diabetes,
Involvement of heat shock factor-1 (HSF1) in upregulation of plasminogen activator Inhlbltor- ' in streptozotocin-induced diabetic , apolipoprotein E-knockout mice and fibroblasts from HSF1knockout mice. RUOZHI ZHAO, TOORU MIZUNO, ARNOLD LECKSROM, MOHAMMED MOGHADASAIN, GARRY X. SHEN' . Departmens of Internal Medicine , Physiology and Human Nutritional Sciences, University of Manitoba. Winnipeg, MB Plasminoge n activator inhibitor-1 (PAI-1) plays an important role in intravascular thrombosis. atherosclerosis and inflammation. Our previous studies demonstrated that HSF1 is involved in glycated LDL-induced PAI-1 production in cultured vascular endothelial cells. The present study examined the expression of PAI-1 and HSF1 in streptozotocin (STZ)-induced diabetic and apolipoprotein E-knockout (apoE-KO) mice. The level of plasma PAI-1 was significantly elevated in STZ-diabetic mice compared to control m ice. The expression of both PAI-1 and HSF1 was significantly greater in heart tissue of STZ-diabetic mice than that in control mice. The level of PAI-1 was significantly higher in plasma of apoE-KO mice than in C57BLl6J control mice. Increased expressions of PAI-1 and HSF1 were detected in heart, aorta and skeletal muscle of apoE-KO mice compared to control mice. The expression and release of PAI-1 was substantially lower in mouse embryo fibroblast (MEF) from HSF1-KO mice than those in MEF from wide-type mice. Oxidized LDL and glycated LDL significantly increased the expression of PAI1 and HSF1 in MEF from wide-type mice compared to that from HSF1-KO mice. The results demonstrate that HSF1 is associated with metabolic stress-induced PAI-1 expression in STZ-diabetic or apoE-KO mice. The expression of HSF1 is requ ired for diabetes-associated LDL-induced PAI-1 production in MEF.
MAHMOUD
ALI
ISMAEL
R EJEAN
Glucose-fed rats is a model of insulin resistance that displays sensory polyneuropathy and hypertension. This study aimed at comparing the beneficial effects of N-Acetyl-L-Cysteine (NAC, antioxidant) and ramipril (angiotensin- I converting enzyme iuhibitor) on tactile and cold allodynia induced by chronic glucose feeding. Impact of these Ireatments was also assessed on hypertension, plasma glucose and insulin concentrations, insulin resistance and kinin 8 1 receptor expression. Male Wistar rats (50-75 g) were given 10% D-glucose in their drinking water for II weeks or tap water (controls). Glucose-fed rats were treated either with NAC ( l glkglday, gavage), ramipril (1 mg/kg/day in drinking water) or no drug during the last five weeks. Glucose feeding for 6 weeks induced a significant increase in systolic blood pressure and hyperglycaemia which was accompanied by tactile and cold allodynia. At II weeks, plasma insulin, insulin resistance (HOMA index), kinin BI receptor mRNA in spinal cord and renal cortex and BI receptor binding sites in spinal cord were enhanced in glucose-ted rats. NAC and ramipril caused a progressive to complete inhibition of tactile and cold allodynia from 6 to II weeks. High systolic blood pressure, hyperinsulin emia, insulin resistance and kinin 8 1 receptor expression were also normali zed or attenuated in g lucose-fed rats by either treatment. Results suggest that chronic treatment with an antioxidant or an ACE inhibitor provides similar beneficial effects on sensory polyneuropathy, hypertension and insulin resistance in glucose-fed rats. Both therapies were associated with a reduction of the expression of the pro-nociceptive klnin 8 1 receptor.
Supported by CDA
I
TALBOT' ,
COUTURE. Dept Physiology , Faculty of Medicine , Universi t,; de Montreal, Qc, Canada
is critical in the initiation of the cellular responses by activating redox-sensitive transcription factor, nuclear factor kappa B (NF-KB). NF-KB activation may lead to cellular structural and functional a lteration via a ltered expression of cytokines. We investigated the role of NF-KB in the development of early changes in diabetic nephropathy We used a hyperhexosemic, normoinsulinimic model of chronic diabetic complication to avoid potential problems associated with system ic insulin therapy needed for longterm diabetic animals. Hence, NF-KB(P50) knockout mice and their wild-type controls were fed with a 30% galactose diet for 2 months. Renal tissues were analyzed. Galactose-fed animals showed increased in serum reducing sugars, without any alterations of body weight. Wild type galactose-fed animals demonstrated increase in the mRNA expression of ET-I and TGF-~ I. Furthermore ECM proteins, regulated by ET-l and TGF~l, such as fibronectin and collagen I a4 were upregulated. Interestingly, poly ADP ribose polymerase mRNA expression was not altered. Such alterations were prevented in the kidneys of galactose-fed knockout animals. These resu lts demonstrate that early changes in diabetic nephropathy may be mediated through NF-KB-dependent signa ling pathways. Understanding these mechanisms is important in identifying potential novel treatment targets.
31
I Blockade of sensory abnormalities and kinin BI receptor_expression by N-Acetyl-L-Cystcinc and Ramipril in a rat model of insulin resistance
Hyperhexosemia-Induced Vasoactive Factors and Extracellular Matrix Protein Production in Kidneys are Mediated via NF-KB. BRADLEY J. DE SOUZA, JANE CHIU, BIAO FENG, BIJU GEORGE AND SUBRATA CHAKRABARTI , Department of Pathology, Un iversity of Western Ontario, London, ON
32
I
The Thioredoxin System in Diabetic Nephropathy. Andrew Advani*, Darren J. Kelly , Kerri Thai , Renae M. Gow, Robyn G. Langham , Alison J. Cox , Per Knud Christensen, Hans-Henrik Parving , Richard E. Gilbert. Li Ka Shing Knowledge Institute , Toronto, ON; St Vincent 's Hospital, Melbourne, Australia; Steno Denmark; Rigshospitalet, Diabetes Center, Gentofte , Copenhagen, Denmark. Excessive reactive oxygen species (ROS) playa key role in the While traditionally pathogenesis of diabetic nephropathy. viewed as arising from the increased metabolic flux of diabetes , we explored the alternative hypothesis that a down regulation of anti-oxidant systems may also contribute. Injurious oxidized sulphydryl groups can be enzymatically repaired by thiol-reducing systems including the thioredoxin system. Accordingly , we determined the expression of thioredoxin (Trx) and its endogenous inhibitor thioredoxin interacting protein (TxnIP) in vitro , in diabetic rats and in human biopsies. The effects of siRNA -mediated attenuation of high glucose-induced TxnIP over-expression were also assessed. When compared with controls, the kidneys of diabetic rats demonstrated a 2-fold increase in TxnlP mRNA (p
I 309
ORAL PRESENTATIONS
29
I
30
SEBASTIE N
Increase in reactive oxygen species production, in diabetes,
Involvement of heat shock factor-1 (HSF1) in upregulation of plasminogen activator Inhlbltor- ' in streptozotocin-induced diabetic , apolipoprotein E-knockout mice and fibroblasts from HSF1knockout mice. RUOZHI ZHAO, TOORU MIZUNO, ARNOLD LECKSROM, MOHAMMED MOGHADASAIN, GARRY X. SHEN' . Departmens of Internal Medicine , Physiology and Human Nutritional Sciences, University of Manitoba. Winnipeg, MB Plasminoge n activator inhibitor-1 (PAI-1) plays an important role in intravascular thrombosis. atherosclerosis and inflammation. Our previous studies demonstrated that HSF1 is involved in glycated LDL-induced PAI-1 production in cultured vascular endothelial cells. The present study examined the expression of PAI-1 and HSF1 in streptozotocin (STZ)-induced diabetic and apolipoprotein E-knockout (apoE-KO) mice. The level of plasma PAI-1 was significantly elevated in STZ-diabetic mice compared to control m ice. The expression of both PAI-1 and HSF1 was significantly greater in heart tissue of STZ-diabetic mice than that in control mice. The level of PAI-1 was significantly higher in plasma of apoE-KO mice than in C57BLl6J control mice. Increased expressions of PAI-1 and HSF1 were detected in heart, aorta and skeletal muscle of apoE-KO mice compared to control mice. The expression and release of PAI-1 was substantially lower in mouse embryo fibroblast (MEF) from HSF1-KO mice than those in MEF from wide-type mice. Oxidized LDL and glycated LDL significantly increased the expression of PAI1 and HSF1 in MEF from wide-type mice compared to that from HSF1-KO mice. The results demonstrate that HSF1 is associated with metabolic stress-induced PAI-1 expression in STZ-diabetic or apoE-KO mice. The expression of HSF1 is requ ired for diabetes-associated LDL-induced PAI-1 production in MEF.
MAHMOUD
ALI
ISMAEL
R EJEAN
Glucose-fed rats is a model of insulin resistance that displays sensory polyneuropathy and hypertension. This study aimed at comparing the beneficial effects of N-Acetyl-L-Cysteine (NAC, antioxidant) and ramipril (angiotensin- I converting enzyme iuhibitor) on tactile and cold allodynia induced by chronic glucose feeding. Impact of these Ireatments was also assessed on hypertension, plasma glucose and insulin concentrations, insulin resistance and kinin 8 1 receptor expression. Male Wistar rats (50-75 g) were given 10% D-glucose in their drinking water for II weeks or tap water (controls). Glucose-fed rats were treated either with NAC ( l glkglday, gavage), ramipril (1 mg/kg/day in drinking water) or no drug during the last five weeks. Glucose feeding for 6 weeks induced a significant increase in systolic blood pressure and hyperglycaemia which was accompanied by tactile and cold allodynia. At II weeks, plasma insulin, insulin resistance (HOMA index), kinin BI receptor mRNA in spinal cord and renal cortex and BI receptor binding sites in spinal cord were enhanced in glucose-ted rats. NAC and ramipril caused a progressive to complete inhibition of tactile and cold allodynia from 6 to II weeks. High systolic blood pressure, hyperinsulin emia, insulin resistance and kinin 8 1 receptor expression were also normali zed or attenuated in g lucose-fed rats by either treatment. Results suggest that chronic treatment with an antioxidant or an ACE inhibitor provides similar beneficial effects on sensory polyneuropathy, hypertension and insulin resistance in glucose-fed rats. Both therapies were associated with a reduction of the expression of the pro-nociceptive klnin 8 1 receptor.
Supported by CDA
I
TALBOT' ,
COUTURE. Dept Physiology , Faculty of Medicine , Universi t,; de Montreal, Qc, Canada
is critical in the initiation of the cellular responses by activating redox-sensitive transcription factor, nuclear factor kappa B (NF-KB). NF-KB activation may lead to cellular structural and functional a lteration via a ltered expression of cytokines. We investigated the role of NF-KB in the development of early changes in diabetic nephropathy We used a hyperhexosemic, normoinsulinimic model of chronic diabetic complication to avoid potential problems associated with system ic insulin therapy needed for longterm diabetic animals. Hence, NF-KB(P50) knockout mice and their wild-type controls were fed with a 30% galactose diet for 2 months. Renal tissues were analyzed. Galactose-fed animals showed increased in serum reducing sugars, without any alterations of body weight. Wild type galactose-fed animals demonstrated increase in the mRNA expression of ET-I and TGF-~ I. Furthermore ECM proteins, regulated by ET-l and TGF~l, such as fibronectin and collagen I a4 were upregulated. Interestingly, poly ADP ribose polymerase mRNA expression was not altered. Such alterations were prevented in the kidneys of galactose-fed knockout animals. These resu lts demonstrate that early changes in diabetic nephropathy may be mediated through NF-KB-dependent signa ling pathways. Understanding these mechanisms is important in identifying potential novel treatment targets.
31
I Blockade of sensory abnormalities and kinin BI receptor_expression by N-Acetyl-L-Cystcinc and Ramipril in a rat model of insulin resistance
Hyperhexosemia-Induced Vasoactive Factors and Extracellular Matrix Protein Production in Kidneys are Mediated via NF-KB. BRADLEY J. DE SOUZA, JANE CHIU, BIAO FENG, BIJU GEORGE AND SUBRATA CHAKRABARTI , Department of Pathology, Un iversity of Western Ontario, London, ON
32
I
The Thioredoxin System in Diabetic Nephropathy. Andrew Advani*, Darren J. Kelly , Kerri Thai , Renae M. Gow, Robyn G. Langham , Alison J. Cox , Per Knud Christensen, Hans-Henrik Parving , Richard E. Gilbert. Li Ka Shing Knowledge Institute , Toronto, ON; St Vincent 's Hospital, Melbourne, Australia; Steno Denmark; Rigshospitalet, Diabetes Center, Gentofte , Copenhagen, Denmark. Excessive reactive oxygen species (ROS) playa key role in the While traditionally pathogenesis of diabetic nephropathy. viewed as arising from the increased metabolic flux of diabetes , we explored the alternative hypothesis that a down regulation of anti-oxidant systems may also contribute. Injurious oxidized sulphydryl groups can be enzymatically repaired by thiol-reducing systems including the thioredoxin system. Accordingly , we determined the expression of thioredoxin (Trx) and its endogenous inhibitor thioredoxin interacting protein (TxnIP) in vitro , in diabetic rats and in human biopsies. The effects of siRNA -mediated attenuation of high glucose-induced TxnIP over-expression were also assessed. When compared with controls, the kidneys of diabetic rats demonstrated a 2-fold increase in TxnlP mRNA (p
I 309