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Glucose amplifies fatty acid-induced endoplasmic reticulum stress in pancreatic beta-cells via activation of mTORC1.

Bachar E, Ariav Y, Ketzinel-Gilad M, Cerasi E, Kaiser N, Leibowitz G - PLoS ONE (2009)

Bottom Line: We found that glucose amplifies palmitate-induced ER stress by increasing IRE1alpha protein levels and activating the JNK pathway, leading to increased beta-cell apoptosis.Finally, we found that JNK inhibition decreased beta-cell apoptosis under conditions of glucolipotoxicity.Moreover, in stressed beta-cells mTORC1 inhibition decreases IRE1alpha protein expression and JNK activity without affecting ER protein load, suggesting that mTORC1 regulates the beta-cell stress response to glucose and fatty acids by modulating the synthesis and activity of specific proteins involved in the execution of the ER stress response.

View Article: PubMed Central - PubMed

Affiliation: Endocrinology and Metabolism Service, Department of Medicine, Hadassah--Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT

Background: Palmitate is a potent inducer of endoplasmic reticulum (ER) stress in beta-cells. In type 2 diabetes, glucose amplifies fatty-acid toxicity for pancreatic beta-cells, leading to beta-cell dysfunction and death. Why glucose exacerbates beta-cell lipotoxicity is largely unknown. Glucose stimulates mTORC1, an important nutrient sensor involved in the regulation of cellular stress. Our study tested the hypothesis that glucose augments lipotoxicity by stimulating mTORC1 leading to increased beta-cell ER stress.

Principal findings: We found that glucose amplifies palmitate-induced ER stress by increasing IRE1alpha protein levels and activating the JNK pathway, leading to increased beta-cell apoptosis. Moreover, glucose increased mTORC1 activity and its inhibition by rapamycin decreased beta-cell apoptosis under conditions of glucolipotoxicity. Inhibition of mTORC1 by rapamycin did not affect proinsulin and total protein synthesis in beta-cells incubated at high glucose with palmitate. However, it decreased IRE1alpha expression and signaling and inhibited JNK pathway activation. In TSC2-deficient mouse embryonic fibroblasts, in which mTORC1 is constitutively active, mTORC1 regulated the stimulation of JNK by ER stressors, but not in response to anisomycin, which activates JNK independent of ER stress. Finally, we found that JNK inhibition decreased beta-cell apoptosis under conditions of glucolipotoxicity.

Conclusions/significance: Collectively, our findings suggest that mTORC1 mediates glucose amplification of lipotoxicity, acting through activation of ER stress and JNK. Thus, mTORC1 is an important transducer of ER stress in beta-cell glucolipotoxicity. Moreover, in stressed beta-cells mTORC1 inhibition decreases IRE1alpha protein expression and JNK activity without affecting ER protein load, suggesting that mTORC1 regulates the beta-cell stress response to glucose and fatty acids by modulating the synthesis and activity of specific proteins involved in the execution of the ER stress response. This novel paradigm may have important implications for understanding beta-cell failure in type 2 diabetes.

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Effects of glucose, palmitate and rapamycin on total protein and proinsulin biosynthesis.INS-1E cells were incubated for 16 h at 3.3 and 22.2 mmol/l glucose with 0.5% BSA with and without 0.5 mmol/l palmitate and 50 nmol/l rapamycin. The last 2 h of the incubations were performed in KRBH-BSA buffer containing similar treatments and 10 µCi L-[2, 3, 4, 5-3H]leucine. After a 2-h incubation at 37°C, leucine incorporation was terminated by ice-cold wash-out in glucose-free KRBH-BSA buffer. Total protein synthesis (A) was determined by trichloroacetic acid precipitation. Proinsulin (PI) biosynthesis (B) was determined by immunoprecipitation with anti-insulin serum. Results are expressed as means±SE of 3 individual experiments, each performed in triplicates. * p<0.05, ** p<0.01, ∧ p<0.001 for the difference between the indicated groups or between the indicated groups and untreated controls at the same glucose concentration.
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pone-0004954-g005: Effects of glucose, palmitate and rapamycin on total protein and proinsulin biosynthesis.INS-1E cells were incubated for 16 h at 3.3 and 22.2 mmol/l glucose with 0.5% BSA with and without 0.5 mmol/l palmitate and 50 nmol/l rapamycin. The last 2 h of the incubations were performed in KRBH-BSA buffer containing similar treatments and 10 µCi L-[2, 3, 4, 5-3H]leucine. After a 2-h incubation at 37°C, leucine incorporation was terminated by ice-cold wash-out in glucose-free KRBH-BSA buffer. Total protein synthesis (A) was determined by trichloroacetic acid precipitation. Proinsulin (PI) biosynthesis (B) was determined by immunoprecipitation with anti-insulin serum. Results are expressed as means±SE of 3 individual experiments, each performed in triplicates. * p<0.05, ** p<0.01, ∧ p<0.001 for the difference between the indicated groups or between the indicated groups and untreated controls at the same glucose concentration.

Mentions: Activation of mTORC1 augments protein synthesis, which may increase the ER client protein load, leading to exacerbation of ER stress. In β-cells, proinsulin is the most abundant protein delivered to the ER. Therefore, we studied the effects of glucose, palmitate and rapamycin on proinsulin and global protein synthesis in β-cells (Figure 5–6). Culture of INS-1E cells at 22.2 mmol/l glucose induced 1.5- and 1.7-fold increase in total protein and proinsulin biosynthesis, respectively. Strikingly, palmitate completely abolished the glucose stimulation of proinsulin and total protein biosynthesis, indicating that in β-cells the induction of ER stress by palmitate shuts off protein synthesis. Rapamycin decreased glucose-stimulated proinsulin and total protein biosynthesis only by 10% without affecting protein synthesis in palmitate-treated INS-1E cells.


Glucose amplifies fatty acid-induced endoplasmic reticulum stress in pancreatic beta-cells via activation of mTORC1.

Bachar E, Ariav Y, Ketzinel-Gilad M, Cerasi E, Kaiser N, Leibowitz G - PLoS ONE (2009)

Effects of glucose, palmitate and rapamycin on total protein and proinsulin biosynthesis.INS-1E cells were incubated for 16 h at 3.3 and 22.2 mmol/l glucose with 0.5% BSA with and without 0.5 mmol/l palmitate and 50 nmol/l rapamycin. The last 2 h of the incubations were performed in KRBH-BSA buffer containing similar treatments and 10 µCi L-[2, 3, 4, 5-3H]leucine. After a 2-h incubation at 37°C, leucine incorporation was terminated by ice-cold wash-out in glucose-free KRBH-BSA buffer. Total protein synthesis (A) was determined by trichloroacetic acid precipitation. Proinsulin (PI) biosynthesis (B) was determined by immunoprecipitation with anti-insulin serum. Results are expressed as means±SE of 3 individual experiments, each performed in triplicates. * p<0.05, ** p<0.01, ∧ p<0.001 for the difference between the indicated groups or between the indicated groups and untreated controls at the same glucose concentration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654723&req=5

pone-0004954-g005: Effects of glucose, palmitate and rapamycin on total protein and proinsulin biosynthesis.INS-1E cells were incubated for 16 h at 3.3 and 22.2 mmol/l glucose with 0.5% BSA with and without 0.5 mmol/l palmitate and 50 nmol/l rapamycin. The last 2 h of the incubations were performed in KRBH-BSA buffer containing similar treatments and 10 µCi L-[2, 3, 4, 5-3H]leucine. After a 2-h incubation at 37°C, leucine incorporation was terminated by ice-cold wash-out in glucose-free KRBH-BSA buffer. Total protein synthesis (A) was determined by trichloroacetic acid precipitation. Proinsulin (PI) biosynthesis (B) was determined by immunoprecipitation with anti-insulin serum. Results are expressed as means±SE of 3 individual experiments, each performed in triplicates. * p<0.05, ** p<0.01, ∧ p<0.001 for the difference between the indicated groups or between the indicated groups and untreated controls at the same glucose concentration.
Mentions: Activation of mTORC1 augments protein synthesis, which may increase the ER client protein load, leading to exacerbation of ER stress. In β-cells, proinsulin is the most abundant protein delivered to the ER. Therefore, we studied the effects of glucose, palmitate and rapamycin on proinsulin and global protein synthesis in β-cells (Figure 5–6). Culture of INS-1E cells at 22.2 mmol/l glucose induced 1.5- and 1.7-fold increase in total protein and proinsulin biosynthesis, respectively. Strikingly, palmitate completely abolished the glucose stimulation of proinsulin and total protein biosynthesis, indicating that in β-cells the induction of ER stress by palmitate shuts off protein synthesis. Rapamycin decreased glucose-stimulated proinsulin and total protein biosynthesis only by 10% without affecting protein synthesis in palmitate-treated INS-1E cells.

Bottom Line: We found that glucose amplifies palmitate-induced ER stress by increasing IRE1alpha protein levels and activating the JNK pathway, leading to increased beta-cell apoptosis.Finally, we found that JNK inhibition decreased beta-cell apoptosis under conditions of glucolipotoxicity.Moreover, in stressed beta-cells mTORC1 inhibition decreases IRE1alpha protein expression and JNK activity without affecting ER protein load, suggesting that mTORC1 regulates the beta-cell stress response to glucose and fatty acids by modulating the synthesis and activity of specific proteins involved in the execution of the ER stress response.

View Article: PubMed Central - PubMed

Affiliation: Endocrinology and Metabolism Service, Department of Medicine, Hadassah--Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT

Background: Palmitate is a potent inducer of endoplasmic reticulum (ER) stress in beta-cells. In type 2 diabetes, glucose amplifies fatty-acid toxicity for pancreatic beta-cells, leading to beta-cell dysfunction and death. Why glucose exacerbates beta-cell lipotoxicity is largely unknown. Glucose stimulates mTORC1, an important nutrient sensor involved in the regulation of cellular stress. Our study tested the hypothesis that glucose augments lipotoxicity by stimulating mTORC1 leading to increased beta-cell ER stress.

Principal findings: We found that glucose amplifies palmitate-induced ER stress by increasing IRE1alpha protein levels and activating the JNK pathway, leading to increased beta-cell apoptosis. Moreover, glucose increased mTORC1 activity and its inhibition by rapamycin decreased beta-cell apoptosis under conditions of glucolipotoxicity. Inhibition of mTORC1 by rapamycin did not affect proinsulin and total protein synthesis in beta-cells incubated at high glucose with palmitate. However, it decreased IRE1alpha expression and signaling and inhibited JNK pathway activation. In TSC2-deficient mouse embryonic fibroblasts, in which mTORC1 is constitutively active, mTORC1 regulated the stimulation of JNK by ER stressors, but not in response to anisomycin, which activates JNK independent of ER stress. Finally, we found that JNK inhibition decreased beta-cell apoptosis under conditions of glucolipotoxicity.

Conclusions/significance: Collectively, our findings suggest that mTORC1 mediates glucose amplification of lipotoxicity, acting through activation of ER stress and JNK. Thus, mTORC1 is an important transducer of ER stress in beta-cell glucolipotoxicity. Moreover, in stressed beta-cells mTORC1 inhibition decreases IRE1alpha protein expression and JNK activity without affecting ER protein load, suggesting that mTORC1 regulates the beta-cell stress response to glucose and fatty acids by modulating the synthesis and activity of specific proteins involved in the execution of the ER stress response. This novel paradigm may have important implications for understanding beta-cell failure in type 2 diabetes.

Show MeSH
Related in: MedlinePlus