Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes
ABSTRACT: Endoplasmic reticulum (ER) stress can be induced by various stimuli and triggers the unfolded protein response to activate intracellular signaling pathways that are mediated by 3 ER-resident sensors: inositol requiring protein-1α (IRE1α), PKR-like ER kinase (PERK), and activating transcrip...
| Principais autores: | , , , , , , , , , , , |
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| Formato: | Artigo |
| Idioma: | English |
| Publicado em: |
Elsevier
2022-08-01
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| coleção: | Journal of Dairy Science |
| Assuntos: | |
| Acesso em linha: | http://www.sciencedirect.com/science/article/pii/S0022030222003770 |
| _version_ | 1828785066194501632 |
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| author | Zhiyuan Fang Wenwen Gao Qianming Jiang Juan J. Loor Chenchen Zhao Xiliang Du Min Zhang Yuxiang Song Zhe Wang Guowen Liu Xinwei Li Lin Lei |
| author_facet | Zhiyuan Fang Wenwen Gao Qianming Jiang Juan J. Loor Chenchen Zhao Xiliang Du Min Zhang Yuxiang Song Zhe Wang Guowen Liu Xinwei Li Lin Lei |
| author_sort | Zhiyuan Fang |
| collection | DOAJ |
| description | ABSTRACT: Endoplasmic reticulum (ER) stress can be induced by various stimuli and triggers the unfolded protein response to activate intracellular signaling pathways that are mediated by 3 ER-resident sensors: inositol requiring protein-1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6). In nonruminants, ER stress plays a critical role in hepatic insulin resistance. However, whether ER stress plays a role in nonesterified fatty acid (NEFA)-induced hepatic insulin resistance in dairy cows is still unknown. Experiments were conducted using primary bovine hepatocytes isolated from 5 healthy calves (body weight: 30–40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h. Treatment with NEFA elevated abundance of phosphorylated IRE1α and PERK, and cleavage of ATF6, along with the ER stress-associated genes XBP1, ATF4, and DNAJC3, resulting in both linear and quadratic effects. Furthermore, ER Tracker red staining and transmission electron microscopy results indicated that ER was dilated and degranulated in response to NEFA treatment, suggesting that ER stress was induced by NEFA treatment in bovine hepatocytes. Second, to assess the effect of ER stress on NEFA-induced insulin resistance, hepatocytes were treated with different concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 5 h with or without tauroursodeoxycholic acid (TUDCA, a canonical inhibitor of ER stress). Here, NEFA induced insulin resistance by increasing the abundance of insulin receptor substrate-1 (IRS1) phosphorylation at the inhibitory residue Ser 307 (S307) and decreasing the abundance of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) in a dose-dependent manner. This was accompanied by upregulation of an abundance of gluconeogenic genes [phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6-Pase)]. These detrimental effects of NEFA on insulin signaling could be reversed with TUDCA treatment, indicating a mechanistic link between ER stress and NEFA-induced insulin resistance. In a third experiment, pGPU6/GFP/Neo vectors containing short hairpin RNA targeting IRE1α were used to silence IRE1α transcription, and GSK2656157 (PERK phosphorylation inhibitor) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF; an inhibitor of ATF6) were used to block PERK and ATF6 branches, respectively. Notably, the silencing of the IRE1α branch improved NEFA-induced insulin resistance by decreasing phosphorylation of IRS1 (S307) and increasing phosphorylation of AKT and GSK3β, and reducing PEPCK and G6-Pase mRNA abundance, which was likely dependent on IRE1α kinase activity. Similarly, blockage of the PERK branch increased phosphorylation of AKT and GSK3β, and reduced PEPCK and G6-Pase mRNA abundance, but had no effect on phosphorylation of IRS1 (S307). However, results showed that inhibition of the ATF6 branch had no effects on phosphorylation of IRS1, AKT, and GSK3β, and instead found increasing PEPCK and G6-Pase mRNA abundance. Taken together, data in the present study found that impeding IRE1α and PERK signaling might aid in relieving hepatic insulin resistance. However, the more detailed mechanisms of how IRE1α and PERK signaling contribute to hepatic insulin resistance in dairy cows remain to be determined. |
| first_indexed | 2024-12-11T23:43:07Z |
| format | Article |
| id | doaj.art-dc366ec1b9f843dea6268c63967d4fe2 |
| institution | Directory Open Access Journal |
| issn | 0022-0302 |
| language | English |
| last_indexed | 2024-12-11T23:43:07Z |
| publishDate | 2022-08-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Dairy Science |
| spelling | doaj.art-dc366ec1b9f843dea6268c63967d4fe22022-12-22T00:45:41ZengElsevierJournal of Dairy Science0022-03022022-08-01105868956908Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytesZhiyuan Fang0Wenwen Gao1Qianming Jiang2Juan J. Loor3Chenchen Zhao4Xiliang Du5Min Zhang6Yuxiang Song7Zhe Wang8Guowen Liu9Xinwei Li10Lin Lei11State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, ChinaState Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, ChinaMammalian NutriPhysioGenomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana 61801Mammalian NutriPhysioGenomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana 61801State Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, ChinaState Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, ChinaState Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, ChinaState Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, ChinaState Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, ChinaState Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, ChinaState Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, ChinaState Key Laboratory for Zoonotic Diseases, Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, Jilin, China; Corresponding authorABSTRACT: Endoplasmic reticulum (ER) stress can be induced by various stimuli and triggers the unfolded protein response to activate intracellular signaling pathways that are mediated by 3 ER-resident sensors: inositol requiring protein-1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6). In nonruminants, ER stress plays a critical role in hepatic insulin resistance. However, whether ER stress plays a role in nonesterified fatty acid (NEFA)-induced hepatic insulin resistance in dairy cows is still unknown. Experiments were conducted using primary bovine hepatocytes isolated from 5 healthy calves (body weight: 30–40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h. Treatment with NEFA elevated abundance of phosphorylated IRE1α and PERK, and cleavage of ATF6, along with the ER stress-associated genes XBP1, ATF4, and DNAJC3, resulting in both linear and quadratic effects. Furthermore, ER Tracker red staining and transmission electron microscopy results indicated that ER was dilated and degranulated in response to NEFA treatment, suggesting that ER stress was induced by NEFA treatment in bovine hepatocytes. Second, to assess the effect of ER stress on NEFA-induced insulin resistance, hepatocytes were treated with different concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 5 h with or without tauroursodeoxycholic acid (TUDCA, a canonical inhibitor of ER stress). Here, NEFA induced insulin resistance by increasing the abundance of insulin receptor substrate-1 (IRS1) phosphorylation at the inhibitory residue Ser 307 (S307) and decreasing the abundance of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) in a dose-dependent manner. This was accompanied by upregulation of an abundance of gluconeogenic genes [phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6-Pase)]. These detrimental effects of NEFA on insulin signaling could be reversed with TUDCA treatment, indicating a mechanistic link between ER stress and NEFA-induced insulin resistance. In a third experiment, pGPU6/GFP/Neo vectors containing short hairpin RNA targeting IRE1α were used to silence IRE1α transcription, and GSK2656157 (PERK phosphorylation inhibitor) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF; an inhibitor of ATF6) were used to block PERK and ATF6 branches, respectively. Notably, the silencing of the IRE1α branch improved NEFA-induced insulin resistance by decreasing phosphorylation of IRS1 (S307) and increasing phosphorylation of AKT and GSK3β, and reducing PEPCK and G6-Pase mRNA abundance, which was likely dependent on IRE1α kinase activity. Similarly, blockage of the PERK branch increased phosphorylation of AKT and GSK3β, and reduced PEPCK and G6-Pase mRNA abundance, but had no effect on phosphorylation of IRS1 (S307). However, results showed that inhibition of the ATF6 branch had no effects on phosphorylation of IRS1, AKT, and GSK3β, and instead found increasing PEPCK and G6-Pase mRNA abundance. Taken together, data in the present study found that impeding IRE1α and PERK signaling might aid in relieving hepatic insulin resistance. However, the more detailed mechanisms of how IRE1α and PERK signaling contribute to hepatic insulin resistance in dairy cows remain to be determined.http://www.sciencedirect.com/science/article/pii/S0022030222003770insulin resistancefatty acidsendoplasmic reticulum stressIRE1αPERK |
| spellingShingle | Zhiyuan Fang Wenwen Gao Qianming Jiang Juan J. Loor Chenchen Zhao Xiliang Du Min Zhang Yuxiang Song Zhe Wang Guowen Liu Xinwei Li Lin Lei Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes Journal of Dairy Science insulin resistance fatty acids endoplasmic reticulum stress IRE1α PERK |
| title | Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes |
| title_full | Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes |
| title_fullStr | Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes |
| title_full_unstemmed | Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes |
| title_short | Targeting IRE1α and PERK in the endoplasmic reticulum stress pathway attenuates fatty acid-induced insulin resistance in bovine hepatocytes |
| title_sort | targeting ire1α and perk in the endoplasmic reticulum stress pathway attenuates fatty acid induced insulin resistance in bovine hepatocytes |
| topic | insulin resistance fatty acids endoplasmic reticulum stress IRE1α PERK |
| url | http://www.sciencedirect.com/science/article/pii/S0022030222003770 |
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