- Email: [email protected]
Neu Mammary Carcinogenesis
MONTREAL 2008 ABSTRACTS
| 333
ABSTRACT #183 C&SS COMPLICATIONS
POSTER PRESENTATIONS 120 Modulation of Retinal Blood Flow in Type 1 Diabetic Rats by Kinins B1 Receptor. MYLÈNE POULIOT*, RÉJEAN COUTURE, ELVIRE VAUCHER. School of Optometry and Department of Physiology, Université de Montréal, Montreal, QC, Canada. Diabetes is associated with pathological microvascular changes in the retina caused by oxidative stress and inÀammatory mechanisms in response to hyperglycemia. Recent studies suggest that the inducible B1 receptor (B1R) is overexpressed in retinal microvessels of Streptozotocin-diabetic rats. The activation of B1R dilates retinal microvessels in vitro. Moreover, B1R enhances vascular permeability in this model, a pathologic feature of diabetic retinopathy. The objective of this study was to determine whether B1R could modulate retinal blood Àow in type 1 diabetes. Male Wistar rats (n=20) weighing 200-225g were rendered diabetic with a single i.p. injection of Streptozotocin (STZ, 65 mg/kg). Agematched controls received sodium citrate buffer vehicle. On the fourth day post-STZ injection, the B1R antagonist SSR240612 (Sano¿Aventis) was administrated orally (10 mg/kg) 3 hours prior to blood Àow measurement. Retinal perfusion was measured in awake animals by autoradiography using N-isopropyl-p-14C-iodoamphetamine (14CIMP, 100 ȝCi/kg, i.v. injection) as a tracer. The rats were sacri¿ced two minutes after 14C-IMP injection, the eyes were enucleated and both retinas were dissected out. One retina was digested and radioactivity content was determined with a scintillation counter. The other retina was whole-mounted on a glass slide and exposed with brain sections on X-ray ¿lm for the quanti¿cation of regional changes with computerized analysis (MCID basic 7.0). Retinal blood Àow was slightly but not signi¿cantly decreased in STZ-diabetic rats (80±11 ml/100g/min) compared to control rats (92±18 ml/100g/min). The treatment with B1R antagonist signi¿cantly reduced retinal blood Àow in STZ-diabetic rats (72±9 ml/100g/min) in comparison to control animals (p=0.037, Bonferonni post-hoc test). B1R appeared to have a protective role on retinal vasculature by preventing a strong decrease of retinal blood Àow in diabetic rats. Supported by FRSQ Vision Network and CIHR.
122
Antidiabetic effects of Nigella sativa are mediated by activation of insulin and AMPK pathways, and by mitochondrial uncoupling. BENHADDOUANDALOUSSI ALI*, MARTINEAU C. LOUIS, VALLERAND DIANE, HADDAD YARA, HADDAD S. PIERRE. Natural Health Products and Metabolic Diseases Laboratory, Department of Pharmacology, Université de Montréal, Montréal, QC. Nigella sativa (NS) or black seed is a plant widely used in traditional medicine of north African countries. During the last decade, several studies have shown that extracts from the seeds of NS have an antidiabetic effect. However, the intracellular mechanism of action through which the plant exerts its effect is still unknown. The purpose of our study was to determine the effect of NS on the main signalling pathways involved in the regulation of glucose homeostasis and metabolism, as well as on mitochondrial function in insulin-sensitive cells. C2C12 myotubes, hepatocytes H4IIE and 3T3-L1 adipocytes were treated with ethanolic extract of NS (200 g/ml) for 18 hours. Western blot analysis was carried out to assess key proteins of the insulin (AKT, ERK1/2) and AMPK (AMPK, ACC) pathways. Isolated liver mitochondria were also used to assess the effect of NS respiration and oxidative phosphorylation. NS was found to activate AKT and ERK1/2 in C2C12 to values nearly 50% greater than the vehicle DMSO. Likewise, NS increased phosphorylation of AMPK and ACC in a manner is similar to the positive control AICAR. In H4IIE, NS increased the phosphorylation of AKT, yet reduced the activation of ERK1/2. The hepatic AMPK pathway was also stimulated. In contrast, NS remained without effect on either the insulin or the AMPK pathway in 3T3-L1 adipocytes. Finally, NS reduced the oxygen consumption of isolated liver mitochondria in a dose-dependant manner (25 to 200 g/ml). The antidiabetic effect of NS can therefore be attributed to an increased sensitivity to insulin and an enhancement of the insulinindependent AMPK pathway that can be related to inhibitory effects on the mitochondrial respiratory chain. Interesting cell-specific differences in activity also exist. These data reinforce our previous results of glucose transport stimulation by NS (Benhaddou et al, pharmaceutical biology, 2008) and validate the ethnopharmacological use of NS as a treatment for diabetes. Sponsored by CDA and CIHR.
121
ROLE OF THE KININ B1 RECEPTOR (B1R) IN DIABETIC RATS EXPOSED TO SEPTIC SHOCK N EJLA T IDJANE AND R ÉJEAN C OUTURE . Dept Physiology, Faculty of Medicine, Université de Montréal, Qc, Canada Both diabetes and septic shock increase the production of reactive oxygen species and pro-inflammatory cytokines, leading to higher B1R expression. Bacterial lipopolysacharides (LPS) is commonly used to induce septic shock in animal models. Streptozotocin (STZ)-induced diabetes increases the expression of B1R in various peripheral tissues, brain and spinal cord in rats. Our objective was to study the contribution of B1R in the association of the septic shock and diabetes which increases the risk of morbidity and mortality. Sprague-Dawley rats (225-250 g) treated with STZ (65mg/kg, ip) or vehicle received LPS (2 mg / kg, i.a.) or vehicle in the presence or absence of the B1R antagonist, SSR240612 (10 mg/kg), administered by gavage. Body temperature and glycaemia were monitored 0-24h after the treatment. In addition, SSR240612 was administered twice (9 am, 9 pm) and rats were sacrificed the following morning at 9 am after 12 h fasting to measure its impact on various parameters: plasma insulin and glucose, oedema and vascular permeability in various tissues (using Blue Evans), hematocrite and B1R mRNA (real-time PCR) in the renal cortex and heart. The increase in body temperature caused by LPS treated or not with STZ was blocked by SSR240612. The antagonist normalized hyperglycaemia and improved insulin deficiency in STZ rats. SSR240612 inhibited oedema and reduced vascular permeability in all tissues from STZ-diabetics rats treated or not with LPS. The hematocrite was not affected after treatment with LPS or STZ. Treatment with the B1R antagonist also prevented the induction of B1R in the heart and renal cortex in LPS and/or STZ. Data suggest that kinin B1R is induced and overexpressed in diabetes and septic shock. The blockade of B1R with SSR240612 produced anti-inflammatory, anti-pyretic and anti-diabetic effects which improved rat survival. Hence, kinin B1R antagonists can be of therapeutic value in the treatment of septic shock which is exacerbated in diabetes. [Supported by the CDA and CIHR]
123
Evidence for a Tumor Promoting Effect of High Fat Diet in the Absence of Hyperinsulinemia in HER2/Neu Mammary Carcinogenesis. 1 YAEL BABICHEV*; 1SARAH KHALID; 1DAVID HWANG; 1 RANI KOLLI; 1SVETLANA ALTAMENTOVA; 2MICHAEL POLLAK; 3NAHUM SONENBERG; 1I. GEORGE FANTUS 1 Mount Sinai Hospital, Toronto, ON; 2 McGill University, Montreal, QC The mechanism of the association between breast cancer and obesity remains unknown. Hyperinsulinemia and insulin resistance are associated with obesity and have been suggested to contribute to an increased risk of breast cancer and a poor prognosis. To examine the potential role of insulin, mice over-expressing HER2/Neu in the mammary gland (MMTV-HER2/Neu) were fed either a high fat (45% of calories) (HFD) or low fat (10% of calories) (LFD) diet from 13 weeks of age and followed for up to 1 year. There was a significant increase in body weight in mice on HFD (p<0.05), and the HFD mice displayed a markedly greater fat mass determined by MRI (p<0.01). Glucose intolerance was observed from 3 months of age on HFD, but insulin levels were not elevated. Mice on HFD had an earlier onset of a second tumor and a 2-fold greater incidence of multiple tumors, while the time of onset of a first tumor and tumor growth rates were not altered. Immunoblotting of tumor cell lysates from mice injected with either insulin or saline at the time of sacrifice, revealed no change in basal phosphorylation of IRS-1/2, Akt/PKB and p70S6K, while insulin stimulated phosphorylation of these proteins revealed similar maximum levels at an earlier time (5 min) in HFD than in LFD (10 min) mice. In skeletal muscle, a classical metabolic target tissue of insulin, there was decrease in insulin stimulated phosphorylation of signalling proteins in HFD. Immunohistochemistry revealed no difference in staining for the proliferative marker, Ki67, between diets. These findings suggest that HFD, in the absence of hyperinsulinemia, mediates a tumor promoting, but not a tumor growth effect in this model of mammary carcinogenesis.
ABSTRACT #110 C&SS
INSULIN ACTION/RESIST
| 333
ABSTRACT #183 C&SS COMPLICATIONS
POSTER PRESENTATIONS 120 Modulation of Retinal Blood Flow in Type 1 Diabetic Rats by Kinins B1 Receptor. MYLÈNE POULIOT*, RÉJEAN COUTURE, ELVIRE VAUCHER. School of Optometry and Department of Physiology, Université de Montréal, Montreal, QC, Canada. Diabetes is associated with pathological microvascular changes in the retina caused by oxidative stress and inÀammatory mechanisms in response to hyperglycemia. Recent studies suggest that the inducible B1 receptor (B1R) is overexpressed in retinal microvessels of Streptozotocin-diabetic rats. The activation of B1R dilates retinal microvessels in vitro. Moreover, B1R enhances vascular permeability in this model, a pathologic feature of diabetic retinopathy. The objective of this study was to determine whether B1R could modulate retinal blood Àow in type 1 diabetes. Male Wistar rats (n=20) weighing 200-225g were rendered diabetic with a single i.p. injection of Streptozotocin (STZ, 65 mg/kg). Agematched controls received sodium citrate buffer vehicle. On the fourth day post-STZ injection, the B1R antagonist SSR240612 (Sano¿Aventis) was administrated orally (10 mg/kg) 3 hours prior to blood Àow measurement. Retinal perfusion was measured in awake animals by autoradiography using N-isopropyl-p-14C-iodoamphetamine (14CIMP, 100 ȝCi/kg, i.v. injection) as a tracer. The rats were sacri¿ced two minutes after 14C-IMP injection, the eyes were enucleated and both retinas were dissected out. One retina was digested and radioactivity content was determined with a scintillation counter. The other retina was whole-mounted on a glass slide and exposed with brain sections on X-ray ¿lm for the quanti¿cation of regional changes with computerized analysis (MCID basic 7.0). Retinal blood Àow was slightly but not signi¿cantly decreased in STZ-diabetic rats (80±11 ml/100g/min) compared to control rats (92±18 ml/100g/min). The treatment with B1R antagonist signi¿cantly reduced retinal blood Àow in STZ-diabetic rats (72±9 ml/100g/min) in comparison to control animals (p=0.037, Bonferonni post-hoc test). B1R appeared to have a protective role on retinal vasculature by preventing a strong decrease of retinal blood Àow in diabetic rats. Supported by FRSQ Vision Network and CIHR.
122
Antidiabetic effects of Nigella sativa are mediated by activation of insulin and AMPK pathways, and by mitochondrial uncoupling. BENHADDOUANDALOUSSI ALI*, MARTINEAU C. LOUIS, VALLERAND DIANE, HADDAD YARA, HADDAD S. PIERRE. Natural Health Products and Metabolic Diseases Laboratory, Department of Pharmacology, Université de Montréal, Montréal, QC. Nigella sativa (NS) or black seed is a plant widely used in traditional medicine of north African countries. During the last decade, several studies have shown that extracts from the seeds of NS have an antidiabetic effect. However, the intracellular mechanism of action through which the plant exerts its effect is still unknown. The purpose of our study was to determine the effect of NS on the main signalling pathways involved in the regulation of glucose homeostasis and metabolism, as well as on mitochondrial function in insulin-sensitive cells. C2C12 myotubes, hepatocytes H4IIE and 3T3-L1 adipocytes were treated with ethanolic extract of NS (200 g/ml) for 18 hours. Western blot analysis was carried out to assess key proteins of the insulin (AKT, ERK1/2) and AMPK (AMPK, ACC) pathways. Isolated liver mitochondria were also used to assess the effect of NS respiration and oxidative phosphorylation. NS was found to activate AKT and ERK1/2 in C2C12 to values nearly 50% greater than the vehicle DMSO. Likewise, NS increased phosphorylation of AMPK and ACC in a manner is similar to the positive control AICAR. In H4IIE, NS increased the phosphorylation of AKT, yet reduced the activation of ERK1/2. The hepatic AMPK pathway was also stimulated. In contrast, NS remained without effect on either the insulin or the AMPK pathway in 3T3-L1 adipocytes. Finally, NS reduced the oxygen consumption of isolated liver mitochondria in a dose-dependant manner (25 to 200 g/ml). The antidiabetic effect of NS can therefore be attributed to an increased sensitivity to insulin and an enhancement of the insulinindependent AMPK pathway that can be related to inhibitory effects on the mitochondrial respiratory chain. Interesting cell-specific differences in activity also exist. These data reinforce our previous results of glucose transport stimulation by NS (Benhaddou et al, pharmaceutical biology, 2008) and validate the ethnopharmacological use of NS as a treatment for diabetes. Sponsored by CDA and CIHR.
121
ROLE OF THE KININ B1 RECEPTOR (B1R) IN DIABETIC RATS EXPOSED TO SEPTIC SHOCK N EJLA T IDJANE AND R ÉJEAN C OUTURE . Dept Physiology, Faculty of Medicine, Université de Montréal, Qc, Canada Both diabetes and septic shock increase the production of reactive oxygen species and pro-inflammatory cytokines, leading to higher B1R expression. Bacterial lipopolysacharides (LPS) is commonly used to induce septic shock in animal models. Streptozotocin (STZ)-induced diabetes increases the expression of B1R in various peripheral tissues, brain and spinal cord in rats. Our objective was to study the contribution of B1R in the association of the septic shock and diabetes which increases the risk of morbidity and mortality. Sprague-Dawley rats (225-250 g) treated with STZ (65mg/kg, ip) or vehicle received LPS (2 mg / kg, i.a.) or vehicle in the presence or absence of the B1R antagonist, SSR240612 (10 mg/kg), administered by gavage. Body temperature and glycaemia were monitored 0-24h after the treatment. In addition, SSR240612 was administered twice (9 am, 9 pm) and rats were sacrificed the following morning at 9 am after 12 h fasting to measure its impact on various parameters: plasma insulin and glucose, oedema and vascular permeability in various tissues (using Blue Evans), hematocrite and B1R mRNA (real-time PCR) in the renal cortex and heart. The increase in body temperature caused by LPS treated or not with STZ was blocked by SSR240612. The antagonist normalized hyperglycaemia and improved insulin deficiency in STZ rats. SSR240612 inhibited oedema and reduced vascular permeability in all tissues from STZ-diabetics rats treated or not with LPS. The hematocrite was not affected after treatment with LPS or STZ. Treatment with the B1R antagonist also prevented the induction of B1R in the heart and renal cortex in LPS and/or STZ. Data suggest that kinin B1R is induced and overexpressed in diabetes and septic shock. The blockade of B1R with SSR240612 produced anti-inflammatory, anti-pyretic and anti-diabetic effects which improved rat survival. Hence, kinin B1R antagonists can be of therapeutic value in the treatment of septic shock which is exacerbated in diabetes. [Supported by the CDA and CIHR]
123
Evidence for a Tumor Promoting Effect of High Fat Diet in the Absence of Hyperinsulinemia in HER2/Neu Mammary Carcinogenesis. 1 YAEL BABICHEV*; 1SARAH KHALID; 1DAVID HWANG; 1 RANI KOLLI; 1SVETLANA ALTAMENTOVA; 2MICHAEL POLLAK; 3NAHUM SONENBERG; 1I. GEORGE FANTUS 1 Mount Sinai Hospital, Toronto, ON; 2 McGill University, Montreal, QC The mechanism of the association between breast cancer and obesity remains unknown. Hyperinsulinemia and insulin resistance are associated with obesity and have been suggested to contribute to an increased risk of breast cancer and a poor prognosis. To examine the potential role of insulin, mice over-expressing HER2/Neu in the mammary gland (MMTV-HER2/Neu) were fed either a high fat (45% of calories) (HFD) or low fat (10% of calories) (LFD) diet from 13 weeks of age and followed for up to 1 year. There was a significant increase in body weight in mice on HFD (p<0.05), and the HFD mice displayed a markedly greater fat mass determined by MRI (p<0.01). Glucose intolerance was observed from 3 months of age on HFD, but insulin levels were not elevated. Mice on HFD had an earlier onset of a second tumor and a 2-fold greater incidence of multiple tumors, while the time of onset of a first tumor and tumor growth rates were not altered. Immunoblotting of tumor cell lysates from mice injected with either insulin or saline at the time of sacrifice, revealed no change in basal phosphorylation of IRS-1/2, Akt/PKB and p70S6K, while insulin stimulated phosphorylation of these proteins revealed similar maximum levels at an earlier time (5 min) in HFD than in LFD (10 min) mice. In skeletal muscle, a classical metabolic target tissue of insulin, there was decrease in insulin stimulated phosphorylation of signalling proteins in HFD. Immunohistochemistry revealed no difference in staining for the proliferative marker, Ki67, between diets. These findings suggest that HFD, in the absence of hyperinsulinemia, mediates a tumor promoting, but not a tumor growth effect in this model of mammary carcinogenesis.
ABSTRACT #110 C&SS
INSULIN ACTION/RESIST