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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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Effect of mTORC1 inhibition by rapamycin on glucose and palmitate-induced β-cell apoptosis.INS-1E cells were incubated 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 for 16 h. Apoptosis was assessed using the Cell Death ELISAPLUS assay (Roche Diagnostics) (A) and by Western blot for cleaved caspase 3 (B). Results are expressed as means±SE of 4 individual experiments, each performed in triplicates (A). A representative gel of 3 individual experiments showing the expression of cleaved and uncleaved caspase 3 is presented (B). ** p<0.01, ∧ p<0.001 for the difference between the indicated groups.
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pone-0004954-g001: Effect of mTORC1 inhibition by rapamycin on glucose and palmitate-induced β-cell apoptosis.INS-1E cells were incubated 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 for 16 h. Apoptosis was assessed using the Cell Death ELISAPLUS assay (Roche Diagnostics) (A) and by Western blot for cleaved caspase 3 (B). Results are expressed as means±SE of 4 individual experiments, each performed in triplicates (A). A representative gel of 3 individual experiments showing the expression of cleaved and uncleaved caspase 3 is presented (B). ** p<0.01, ∧ p<0.001 for the difference between the indicated groups.

Mentions: INS-1E β-cells were treated with 0.5 mmol/l palmitate at 3.3 and 22.2 mmol/l glucose for 16 h. Apoptosis was quantified using an ELISA assay for cytosolic oligonucleosome content indicative of apoptosis-induced DNA degradation (Figure 1A). At 3.3 mmol/l glucose, palmitate had a small, non-significant effect on β-cell apoptosis; the tendency for apoptosis was 1.4-fold higher than in untreated cells incubated at 0.5% BSA (w/v). Incubation of INS-1E cells at 22.2 mmol/l sensitized the β-cells to the toxic effect of palmitate: the apoptotic rate of INS-1E β-cells treated with palmitate was now 2.1- and 2.9-fold higher than that of palmitate-treated cells at 3.3 mmol/l glucose and untreated controls at 22.2 mmol/l glucose, respectively.


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)

Effect of mTORC1 inhibition by rapamycin on glucose and palmitate-induced β-cell apoptosis.INS-1E cells were incubated 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 for 16 h. Apoptosis was assessed using the Cell Death ELISAPLUS assay (Roche Diagnostics) (A) and by Western blot for cleaved caspase 3 (B). Results are expressed as means±SE of 4 individual experiments, each performed in triplicates (A). A representative gel of 3 individual experiments showing the expression of cleaved and uncleaved caspase 3 is presented (B). ** p<0.01, ∧ p<0.001 for the difference between the indicated groups.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004954-g001: Effect of mTORC1 inhibition by rapamycin on glucose and palmitate-induced β-cell apoptosis.INS-1E cells were incubated 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 for 16 h. Apoptosis was assessed using the Cell Death ELISAPLUS assay (Roche Diagnostics) (A) and by Western blot for cleaved caspase 3 (B). Results are expressed as means±SE of 4 individual experiments, each performed in triplicates (A). A representative gel of 3 individual experiments showing the expression of cleaved and uncleaved caspase 3 is presented (B). ** p<0.01, ∧ p<0.001 for the difference between the indicated groups.
Mentions: INS-1E β-cells were treated with 0.5 mmol/l palmitate at 3.3 and 22.2 mmol/l glucose for 16 h. Apoptosis was quantified using an ELISA assay for cytosolic oligonucleosome content indicative of apoptosis-induced DNA degradation (Figure 1A). At 3.3 mmol/l glucose, palmitate had a small, non-significant effect on β-cell apoptosis; the tendency for apoptosis was 1.4-fold higher than in untreated cells incubated at 0.5% BSA (w/v). Incubation of INS-1E cells at 22.2 mmol/l sensitized the β-cells to the toxic effect of palmitate: the apoptotic rate of INS-1E β-cells treated with palmitate was now 2.1- and 2.9-fold higher than that of palmitate-treated cells at 3.3 mmol/l glucose and untreated controls at 22.2 mmol/l glucose, respectively.

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