- Email: [email protected]
Fatty acids inhibit mitochondrial fusion and autophagy in beta-cells
Abstracts / Atherosclerosis 233 (2014) 326–330
Objectives: To study the association between glucose control during admission, length of admission and in-hospital mortality among diabetes patients admitted to internal medicine departments. Methods: This is a retrospective analysis of patient records. Included in the analysis were patients with the diagnosis of diabetes mellitus upon admission. Electronic medical records were audited, and information regarding reason for admission, presenting symptom, co-morbidities, all glucose levels during admission, Creatinine, length of admission, and death status. Percent of glucose measurements between 110 and 180 mg/dL was calculated for each patient. Patients were considered as having a hypoglycemia event if, at least one glucose level was found to be below 70 mg/dL. Logistic regression modeling was used to verify the parameters affecting the rate of in-hospital mortality. Linear regression models were used to verify the parameters affecting length of admission. Results: Out of the 5274 patients that were discharged during the last quarter of 2009, 1479 (28.0%) had diabetes upon admission. Mean age was 72.0 12.8 years, and 50.0% were males. Co-morbidities were hypertension (65.4%), hyperlipidemia (40.6%), Ischemia heart disease (36.0%), Renal failure (15.1%), Stroke status (8.4%). The most frequent reason for admission was any type of infection (27.0%). The average glucose levels during the admission were 172.3 72.6 mg/dL. Rate of glucose control varied between patients, and only 34.7% had more than 50% of the values between 110 and 180 mg/dL, and 54.8% had more than 30% within the same range. 8.7% of glucose levels were below 70 mg/dL, and 6.0% of the patients had at least 1 level below 70 mg/dL. Average length of admission was 4.7 5.7 days (median 3 days) and 3.5% died. Among patients with at least one hypoglycemia event, rate of inhospital mortality was higher (18.1%) and length of stay was (11.1 days) compared to diabetes patients without hypoglycemia events (3.5% and 4.4 days respectively, p < 0.001 for both). Logistic regression modeling showed that age, a hypoglycemia event and infection (as reason for admission) were independently associated with in-hospital mortality, and the model accurately predicted 97.5% of the cases. We found that in-hospital length of stay was also predicted by age and hypoglycemia events. Rate of glucose control during the admission was not an independent parameter to influence length of admission nor in-hospital mortality rates. Conclusion: In hospital mortality is associated with Age, a hypogelymia event during the admission and infection as a reason for admission. There is no association between the control rate of glucose during admission and in-hospital mortality nor length of admission.
METFORMIN INHIBITS MACROPHAGE CHOLESTEROL BIOSYNTHESIS RATE: POSSIBLE ROLE FOR METFORMIN-INDUCED OXIDATIVE STRESS Marie Koren-Gluzer, Michael Aviram, Tony Hayek The Lipid Research Laboratory, Technion Faculty of Medicine, the Rappaport Family Institute for Research in the Medical Sciences, and Rambam Medical Center, Haifa, Israel The aim of the present study was to analyze the metformin (MF) effect on two cellular atherogenic activities: cholesterol biosynthesis and oxidativestress (OS) as studied in J774A.1 macrophage cell line. MF (2–5 mM) significantly and dose – dependently reduced macrophage cholesterol content and cholesterol biosynthesis rate from acetate, but not from mevalonate, by up to 68% and 71%, respectively. MF inhibitory effect on cholesterol biosynthesis was similar to that of simvastatin. In contrast to the above anti-atherogenic MF effect, MF significantly increased cellular OS as shown by enhancement of reactive oxygen species (ROS) production by up to 70%, and decrement in cellular reduced glutathione (GSH) levels by up to 67%. Macrophage paraoxonase2 (PON2) expression however, increased by MF, by up to 1.5 folds. To overcome the MF oxidation stimulation, macrophages were incubated with MF together with potent dietary antioxidants, i.e. 5 mg GAE/ml of pomegranate juice (PJ) or 30 mM of vitamin E (VE). Both of these potent antioxidants substantially reduced MF-induced OS, and in parallel, abolished MF inhibitory effect on cholesterol biosynthesis rate. We thus conclude that the inhibition of macrophage cholesterol biosynthesis by MF is related, at least in part, to MFinduced OS.
329
UROKINASE-TYPE PLASMINOGEN ACTIVATOR (UPA) INCREASES TRIGLYCERIDE ACCUMULATION IN HEPATOCYTES AND CONTRIBUTES TO THE DEVELOPMENT OF NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD) Nicole Paland, Aviva Lazarovich, Raymond Coleman, Bianca Fuhrman The Lipid Research Laboratory, Technion Faculty of Medicine and Rambam Medical Center, Haifa, Israel Background and aims: The liver is the central organ of fatty acid and triglyceride metabolism. Oxidation and synthesis of fatty acids and triglycerides is under the control of peroxisome-proliferator-activated receptors (PPARa, PPARg). Impairment of these receptors' function contributes to triglyceride accumulation in the liver, leading to NAFLD. The uPA/uPAR system was shown to regulate gene expression in the liver involving PPARg transcriptional activity, and to affect HDL-cholesterol metabolism. However, involvement of the uPA/uPAR system in triglyceride metabolism was not yet shown. In this study we questioned whether the uPA/uPAR system modulates triglyceride metabolism in the liver, and investigated the mechanisms involved in the observed processes. Methods and results: Five C57BL/6 and five uPAR / mice were administered a high fat diet for a period of ten weeks. When C57BL/6 mice were challenged with a high fat diet, lipid droplets accumulated in their liver and liver levels of triglycerides and fatty acids increased significantly by 100%. In contrast, in uPAR / mice no lipid droplets could be seen in the liver, and the liver levels of triglycerides and fatty acids were not significantly changed. In order to elucidate the mechanism of uPA/uPAR-induced triglyceride accumulation in the liver, the hepatocyte cell line Huh7 was incubated for 18 hours at 37 C with increasing concentrations of uPA. uPA dose-dependently increased the cellular triglyceride mass, and this effect resulted, at least in part, from increased de novo triglyceride synthesis mediated by the enzyme diglyceride acyltransferase 2 (DGAT2), one of the two major enzymes of triglyceride anabolism. Furthermore, the amount of free fatty acids was highly upregulated by uPA through activation of the transcription factor SREBP-1. SREBP-1 and the abundance of fatty acids are under the control of PPARa and PPARg. Chemical inhibition of PPARa and activation of PPARg inhibited uPA-stimulation of fatty acid and triglyceride synthesis, suggesting that uPA activates PPARa and deactivates PPARg in order to increase fatty acid and triglyceride synthesis. Furthermore, inhibition of p38, which is upstream of both PPARa and PPARg, led to a decrease of uPA-mediated triglyceride synthesis. Conclusion: The uPA/uPAR system is involved in the metabolism and storage of fatty acids and triglycerides in the liver. The uPA-mediated upregulation of triglyceride synthesis is dependent on DGAT2, and is under the control of p38. This study presents a new biological function of the uPA/uPAR system and might present a new target for an early therapeutic intervention for NAFLD.
FATTY ACIDS INHIBIT MITOCHONDRIAL FUSION AND AUTOPHAGY IN BETA-CELLS Guy Las a, Anthony Molina b, Gilad Twig b, Orian Shirihai a, b a b
Faculty of Biochemical Medicine, Ben Gurion University, Beer Sheva, Israel; Department of Medicine, Boston Univeristy, Boston, USA
One of the causes for the well-documented association between Type-2diabetes and obesity is believed to be the toxic effect of high levels of free fatty acids on pancreatic beta-cells. Indeed, incubation of beta-cells with fatty acids, such as palmitate, suppresses insulin secretion and induces apoptosis. Yet, the mechanism for this phenomenon is still unclear. In the present study we examined the role of mitochondria – central regulators of insulin secretion and apoptosis – in fatty acid-induced toxicity. INS-1 beta-cell line, were incubated in the presence or absence of 0.4 mM palmitate. After 24 h, half of the cells exposed to palmitate died while the surviving cells showed a strongly fragmented mitochondrial morphology as compared to control cells – which displayed a filamentous morphology. To determine the cause for the fragmentation
330
Abstracts / Atherosclerosis 233 (2014) 326–330
a Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; b Weizmann Institute of Science, Rehovot, Israel
inflammation, adipose tissue stress, and insulin resistance remain poorly characterized. Aim: To characterize reversibility dynamics of obesity-induced metabolic and inflammatory processes in fat and liver. Methods: Mice were fed normal chow (NC-10% fat) or a high-fat (HFF-60% fat) diet for 8 w after which the reversibility group (REV) was switched back to NC for 2 w. Results: Whole-body and fat tissues weights were w60% reversed in REV, while the liver weight fully reversed. Fasting glucose, insulin, and insulin-tolerance-test were fully reversed (ITTAUC: 12509 1160, 6873 652 and 7889 924 for HFF, NC and REV, respectively). Glucose-tolerance-test showed near complete reversibility (GTTAUC: 43594 2728,17955 1558, and 22461 1032, respectively, P < 0.05). In response to intra-peritoneal insulin, HFF displayed impaired insulin responsiveness compared to NC. Interestingly, insulin signaling improved to greater extent in livers than in the fat tissues, which remained significantly insulin resistant. Correspondingly, adipose tissue macrophage infiltration (increased 3.5-fold by HFF) was 3-fold higher in REV than NC, though macrophages' lipid content reversed by w50%. Adipose tissue stress signaling showed near-significant improvement. Ex-vivo, liver-derived Hepa-1 cells exhibited no improvement in insulin-stimulated Akt phosphorylation when cultured with adipose explants from REV compared to fat tissue explants from HFF. Conclusion: Reversibility of insulin resistance, adipose inflammation and hepatic steatosis may occur through pathways distinct from their pathogenesis in obesity: While adipose tissue inflammation causally links obesogenic diet to insulin resistance and hepatic steatosis, these parameters reverse before a significant change in adipose tissue macrophage infiltration, or endocrine function of adipose tissue with hepatocytes, are apparent.
Introduction: Reversal of dys-regulated metabolism in obesity is achievable, but the dynamics and mechanisms for reversal of
Keywords: HFF diet, “Insulin resistance”, “Metabolic inflammation”, Fat tissue-liver crosstalk
we measured the rate of fusion between mitochondria. Fatty acids strongly impaired the fusion between mitochondria and decreased the levels of Mitofusin2 (Mfn2), a mitochondrial fusion protein, by half. Similarly, islets isolated from corpses of type-2 diabetic patients, showed a decrease of 50% in Mfn2 levels. To determine the role of Mfn2 decrease in fatty acid-induced toxicity, we generated Mfn2 conditional KO mice. Mice lacking Mfn2 in their beta-cells showed a striking obese phenotype along with impaired glucose tolerance that led to diabetes. Two hours after glucose challenge, the mice lacking Mfn2 showed a glucose blood level twice that of control (304 mg/dl versus 148 mg/dl). Also, similarly to INS1 cells exposed to fatty acids, Mfn2 KO mice, showed accumulation of fragmented damaged mitochondria. To determine why those mitochondria are not cleared from the cytoplasm, we examined autophagy upon exposure to palmitate. Interestingly, the number of autophagosomes (including those containing mitochondria) was increased by palmitate. Yet, protein degradation via autophagy as measured by pulsechase experiments was strongly suppressed, and lysosomes acidity was reduced. We conclude that palmitate induces mitochondrial fragmentation but prevents mitochondrial clearance from beta-cells by impairing lysosomal acidification.
REVERSIBILITY OF THE METABOLIC DERANGEMENT INFLAMMATION INDUCED BY NUTRITIONAL OBESITY Maayan Vatarescu a, Noa Slutsky a, Tal Pecht a, Ori Shapiro b, Tanya Tarnovcki b, Nava Bashan a, Assaf Rudich a
Nov a,
AND
Hagit
Objectives: To study the association between glucose control during admission, length of admission and in-hospital mortality among diabetes patients admitted to internal medicine departments. Methods: This is a retrospective analysis of patient records. Included in the analysis were patients with the diagnosis of diabetes mellitus upon admission. Electronic medical records were audited, and information regarding reason for admission, presenting symptom, co-morbidities, all glucose levels during admission, Creatinine, length of admission, and death status. Percent of glucose measurements between 110 and 180 mg/dL was calculated for each patient. Patients were considered as having a hypoglycemia event if, at least one glucose level was found to be below 70 mg/dL. Logistic regression modeling was used to verify the parameters affecting the rate of in-hospital mortality. Linear regression models were used to verify the parameters affecting length of admission. Results: Out of the 5274 patients that were discharged during the last quarter of 2009, 1479 (28.0%) had diabetes upon admission. Mean age was 72.0 12.8 years, and 50.0% were males. Co-morbidities were hypertension (65.4%), hyperlipidemia (40.6%), Ischemia heart disease (36.0%), Renal failure (15.1%), Stroke status (8.4%). The most frequent reason for admission was any type of infection (27.0%). The average glucose levels during the admission were 172.3 72.6 mg/dL. Rate of glucose control varied between patients, and only 34.7% had more than 50% of the values between 110 and 180 mg/dL, and 54.8% had more than 30% within the same range. 8.7% of glucose levels were below 70 mg/dL, and 6.0% of the patients had at least 1 level below 70 mg/dL. Average length of admission was 4.7 5.7 days (median 3 days) and 3.5% died. Among patients with at least one hypoglycemia event, rate of inhospital mortality was higher (18.1%) and length of stay was (11.1 days) compared to diabetes patients without hypoglycemia events (3.5% and 4.4 days respectively, p < 0.001 for both). Logistic regression modeling showed that age, a hypoglycemia event and infection (as reason for admission) were independently associated with in-hospital mortality, and the model accurately predicted 97.5% of the cases. We found that in-hospital length of stay was also predicted by age and hypoglycemia events. Rate of glucose control during the admission was not an independent parameter to influence length of admission nor in-hospital mortality rates. Conclusion: In hospital mortality is associated with Age, a hypogelymia event during the admission and infection as a reason for admission. There is no association between the control rate of glucose during admission and in-hospital mortality nor length of admission.
METFORMIN INHIBITS MACROPHAGE CHOLESTEROL BIOSYNTHESIS RATE: POSSIBLE ROLE FOR METFORMIN-INDUCED OXIDATIVE STRESS Marie Koren-Gluzer, Michael Aviram, Tony Hayek The Lipid Research Laboratory, Technion Faculty of Medicine, the Rappaport Family Institute for Research in the Medical Sciences, and Rambam Medical Center, Haifa, Israel The aim of the present study was to analyze the metformin (MF) effect on two cellular atherogenic activities: cholesterol biosynthesis and oxidativestress (OS) as studied in J774A.1 macrophage cell line. MF (2–5 mM) significantly and dose – dependently reduced macrophage cholesterol content and cholesterol biosynthesis rate from acetate, but not from mevalonate, by up to 68% and 71%, respectively. MF inhibitory effect on cholesterol biosynthesis was similar to that of simvastatin. In contrast to the above anti-atherogenic MF effect, MF significantly increased cellular OS as shown by enhancement of reactive oxygen species (ROS) production by up to 70%, and decrement in cellular reduced glutathione (GSH) levels by up to 67%. Macrophage paraoxonase2 (PON2) expression however, increased by MF, by up to 1.5 folds. To overcome the MF oxidation stimulation, macrophages were incubated with MF together with potent dietary antioxidants, i.e. 5 mg GAE/ml of pomegranate juice (PJ) or 30 mM of vitamin E (VE). Both of these potent antioxidants substantially reduced MF-induced OS, and in parallel, abolished MF inhibitory effect on cholesterol biosynthesis rate. We thus conclude that the inhibition of macrophage cholesterol biosynthesis by MF is related, at least in part, to MFinduced OS.
329
UROKINASE-TYPE PLASMINOGEN ACTIVATOR (UPA) INCREASES TRIGLYCERIDE ACCUMULATION IN HEPATOCYTES AND CONTRIBUTES TO THE DEVELOPMENT OF NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD) Nicole Paland, Aviva Lazarovich, Raymond Coleman, Bianca Fuhrman The Lipid Research Laboratory, Technion Faculty of Medicine and Rambam Medical Center, Haifa, Israel Background and aims: The liver is the central organ of fatty acid and triglyceride metabolism. Oxidation and synthesis of fatty acids and triglycerides is under the control of peroxisome-proliferator-activated receptors (PPARa, PPARg). Impairment of these receptors' function contributes to triglyceride accumulation in the liver, leading to NAFLD. The uPA/uPAR system was shown to regulate gene expression in the liver involving PPARg transcriptional activity, and to affect HDL-cholesterol metabolism. However, involvement of the uPA/uPAR system in triglyceride metabolism was not yet shown. In this study we questioned whether the uPA/uPAR system modulates triglyceride metabolism in the liver, and investigated the mechanisms involved in the observed processes. Methods and results: Five C57BL/6 and five uPAR / mice were administered a high fat diet for a period of ten weeks. When C57BL/6 mice were challenged with a high fat diet, lipid droplets accumulated in their liver and liver levels of triglycerides and fatty acids increased significantly by 100%. In contrast, in uPAR / mice no lipid droplets could be seen in the liver, and the liver levels of triglycerides and fatty acids were not significantly changed. In order to elucidate the mechanism of uPA/uPAR-induced triglyceride accumulation in the liver, the hepatocyte cell line Huh7 was incubated for 18 hours at 37 C with increasing concentrations of uPA. uPA dose-dependently increased the cellular triglyceride mass, and this effect resulted, at least in part, from increased de novo triglyceride synthesis mediated by the enzyme diglyceride acyltransferase 2 (DGAT2), one of the two major enzymes of triglyceride anabolism. Furthermore, the amount of free fatty acids was highly upregulated by uPA through activation of the transcription factor SREBP-1. SREBP-1 and the abundance of fatty acids are under the control of PPARa and PPARg. Chemical inhibition of PPARa and activation of PPARg inhibited uPA-stimulation of fatty acid and triglyceride synthesis, suggesting that uPA activates PPARa and deactivates PPARg in order to increase fatty acid and triglyceride synthesis. Furthermore, inhibition of p38, which is upstream of both PPARa and PPARg, led to a decrease of uPA-mediated triglyceride synthesis. Conclusion: The uPA/uPAR system is involved in the metabolism and storage of fatty acids and triglycerides in the liver. The uPA-mediated upregulation of triglyceride synthesis is dependent on DGAT2, and is under the control of p38. This study presents a new biological function of the uPA/uPAR system and might present a new target for an early therapeutic intervention for NAFLD.
FATTY ACIDS INHIBIT MITOCHONDRIAL FUSION AND AUTOPHAGY IN BETA-CELLS Guy Las a, Anthony Molina b, Gilad Twig b, Orian Shirihai a, b a b
Faculty of Biochemical Medicine, Ben Gurion University, Beer Sheva, Israel; Department of Medicine, Boston Univeristy, Boston, USA
One of the causes for the well-documented association between Type-2diabetes and obesity is believed to be the toxic effect of high levels of free fatty acids on pancreatic beta-cells. Indeed, incubation of beta-cells with fatty acids, such as palmitate, suppresses insulin secretion and induces apoptosis. Yet, the mechanism for this phenomenon is still unclear. In the present study we examined the role of mitochondria – central regulators of insulin secretion and apoptosis – in fatty acid-induced toxicity. INS-1 beta-cell line, were incubated in the presence or absence of 0.4 mM palmitate. After 24 h, half of the cells exposed to palmitate died while the surviving cells showed a strongly fragmented mitochondrial morphology as compared to control cells – which displayed a filamentous morphology. To determine the cause for the fragmentation
330
Abstracts / Atherosclerosis 233 (2014) 326–330
a Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; b Weizmann Institute of Science, Rehovot, Israel
inflammation, adipose tissue stress, and insulin resistance remain poorly characterized. Aim: To characterize reversibility dynamics of obesity-induced metabolic and inflammatory processes in fat and liver. Methods: Mice were fed normal chow (NC-10% fat) or a high-fat (HFF-60% fat) diet for 8 w after which the reversibility group (REV) was switched back to NC for 2 w. Results: Whole-body and fat tissues weights were w60% reversed in REV, while the liver weight fully reversed. Fasting glucose, insulin, and insulin-tolerance-test were fully reversed (ITTAUC: 12509 1160, 6873 652 and 7889 924 for HFF, NC and REV, respectively). Glucose-tolerance-test showed near complete reversibility (GTTAUC: 43594 2728,17955 1558, and 22461 1032, respectively, P < 0.05). In response to intra-peritoneal insulin, HFF displayed impaired insulin responsiveness compared to NC. Interestingly, insulin signaling improved to greater extent in livers than in the fat tissues, which remained significantly insulin resistant. Correspondingly, adipose tissue macrophage infiltration (increased 3.5-fold by HFF) was 3-fold higher in REV than NC, though macrophages' lipid content reversed by w50%. Adipose tissue stress signaling showed near-significant improvement. Ex-vivo, liver-derived Hepa-1 cells exhibited no improvement in insulin-stimulated Akt phosphorylation when cultured with adipose explants from REV compared to fat tissue explants from HFF. Conclusion: Reversibility of insulin resistance, adipose inflammation and hepatic steatosis may occur through pathways distinct from their pathogenesis in obesity: While adipose tissue inflammation causally links obesogenic diet to insulin resistance and hepatic steatosis, these parameters reverse before a significant change in adipose tissue macrophage infiltration, or endocrine function of adipose tissue with hepatocytes, are apparent.
Introduction: Reversal of dys-regulated metabolism in obesity is achievable, but the dynamics and mechanisms for reversal of
Keywords: HFF diet, “Insulin resistance”, “Metabolic inflammation”, Fat tissue-liver crosstalk
we measured the rate of fusion between mitochondria. Fatty acids strongly impaired the fusion between mitochondria and decreased the levels of Mitofusin2 (Mfn2), a mitochondrial fusion protein, by half. Similarly, islets isolated from corpses of type-2 diabetic patients, showed a decrease of 50% in Mfn2 levels. To determine the role of Mfn2 decrease in fatty acid-induced toxicity, we generated Mfn2 conditional KO mice. Mice lacking Mfn2 in their beta-cells showed a striking obese phenotype along with impaired glucose tolerance that led to diabetes. Two hours after glucose challenge, the mice lacking Mfn2 showed a glucose blood level twice that of control (304 mg/dl versus 148 mg/dl). Also, similarly to INS1 cells exposed to fatty acids, Mfn2 KO mice, showed accumulation of fragmented damaged mitochondria. To determine why those mitochondria are not cleared from the cytoplasm, we examined autophagy upon exposure to palmitate. Interestingly, the number of autophagosomes (including those containing mitochondria) was increased by palmitate. Yet, protein degradation via autophagy as measured by pulsechase experiments was strongly suppressed, and lysosomes acidity was reduced. We conclude that palmitate induces mitochondrial fragmentation but prevents mitochondrial clearance from beta-cells by impairing lysosomal acidification.
REVERSIBILITY OF THE METABOLIC DERANGEMENT INFLAMMATION INDUCED BY NUTRITIONAL OBESITY Maayan Vatarescu a, Noa Slutsky a, Tal Pecht a, Ori Shapiro b, Tanya Tarnovcki b, Nava Bashan a, Assaf Rudich a
Nov a,
AND
Hagit