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Li HY, Wang Y, Ran M, Gao F, Zhu BY, Xiao HY, Xu C. Tacrolimus induces insulin receptor substrate 1 hyperphosphorylation and inhibits mTORc1/S6K1 cascade in HL7702 cells. World J Diabetes 2025; 16:97910. [PMID: 39959267 PMCID: PMC11718479 DOI: 10.4239/wjd.v16.i2.97910] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/12/2024] [Revised: 08/28/2024] [Accepted: 11/13/2024] [Indexed: 12/30/2024] Open
Abstract
BACKGROUND Tacrolimus (FK506) is a key calcineurin inhibitor used to prevent organ transplant rejection and is effective in improving graft survival. However, it is linked to hyperglycemia and insulin resistance, contributing to new-onset diabetes after transplantation and negatively affecting islet function. AIM To study the effects of tacrolimus on the insulin signaling pathway of hepatocytes. METHODS HL7702 cells were treated with different concentrations of tacrolimus (0.1 mg/L, 1 mg/L, 5 mg/L) for 24 hours. The proteins involved in insulin signaling were detected by Western blotting. RESULTS Compared with the control group, phosphorylation of insulin receptor substrate (IRS) 1 at Ser 307 and Ser 323 were increased significantly when the tacrolimus concentration reached 1 and 5 mg/L. Phosphorylation of IRS1 at Ser 1101 was also increased, although not significantly. However, phosphorylation of Ribosomal protein S6 kinase beta-1 at Thr 389 was decreased significantly. The levels of phosphorylated glycogen synthase kinase 3α Ser 21 and Ser 9 were increased. Surprisingly, phosphorylation of glycogen synthase at Ser 641 was increased. There was no significant change in the activity of glycogen phosphorylase. CONCLUSION Tacrolimus has no direct effect on hepatic glucose metabolism, but inhibits IRS1-mediated insulin signaling. This may be one of the underlying mechanisms by which tacrolimus induces insulin resistance.
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Affiliation(s)
- Hao-Yan Li
- Department of Endocrinology, The Third Medical Center of PLA General Hospital, Beijing 100039, China
| | - Yi Wang
- Department of Endocrinology, The Third Medical Center of PLA General Hospital, Beijing 100039, China
| | - Min Ran
- Department of Endocrinology, The Third Medical Center of PLA General Hospital, Beijing 100039, China
| | - Fei Gao
- Department of Endocrinology, The Third Medical Center of PLA General Hospital, Beijing 100039, China
| | - Bo-Yu Zhu
- Department of Endocrinology, The Third Medical Center of PLA General Hospital, Beijing 100039, China
| | - Hai-Ying Xiao
- Department of Endocrinology, The Third Medical Center of PLA General Hospital, Beijing 100039, China
| | - Chun Xu
- Department of Endocrinology, The Third Medical Center of PLA General Hospital, Beijing 100039, China
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Nogueira CL, Arcanjo AF, Lima ME, Moraes B, da Silva RM, Gondim KC, Konnai S, Ramos I, Santos S, Filardy AD, Pinto KG, Vaz Junior IDS, Logullo C. Starvation Metabolism Adaptations in Tick Embryonic Cells BME26. Int J Mol Sci 2024; 26:87. [PMID: 39795947 PMCID: PMC11719990 DOI: 10.3390/ijms26010087] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2024] [Revised: 12/16/2024] [Accepted: 12/20/2024] [Indexed: 01/13/2025] Open
Abstract
Ticks are hematophagous ectoparasites that transmit pathogens and inflict significant economic losses on the cattle industry. Remarkably, they can survive extended periods of starvation in the absence of a host. The primary objective of this study was to investigate the metabolic adaptations that enable the tick Rhipicephalus microplus to endure starvation using the BME26 cell line as a model system. To simulate nutrient deprivation, cells were subjected to starvation conditions by replacing the L-15 culture medium with phosphate-buffered saline (PBS). Our findings show that these tick cells can endure experimental starvation for up to 48 h. The assessment of glycogen levels in starved cells shows a significant decrease, at both the 24 h and 48 h marks. Additionally, upregulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression, along with downregulation of hexokinase (HK) and pyruvate kinase (PK) gene expression, indicated that BME26 cells would prioritize the gluconeogenic pathway over the glycolytic pathway under starvation conditions. Moreover, the transcriptional levels of autophagy-related genes (ATG) were upregulated in response to starvation. Taken together, our findings suggest a potential role for autophagy in supplying substrates for the gluconeogenic pathway in nutrient-deprived tick cells. This work contributes to the understanding of metabolic regulation in R. microplus ticks and offers valuable insights for tick control strategies.
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Affiliation(s)
- Cintia Lopes Nogueira
- Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil; (C.L.N.); (A.F.A.); (M.E.L.); (B.M.); (R.M.d.S.); (I.R.)
| | - Angélica F. Arcanjo
- Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil; (C.L.N.); (A.F.A.); (M.E.L.); (B.M.); (R.M.d.S.); (I.R.)
| | - Maria Elisa Lima
- Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil; (C.L.N.); (A.F.A.); (M.E.L.); (B.M.); (R.M.d.S.); (I.R.)
| | - Bruno Moraes
- Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil; (C.L.N.); (A.F.A.); (M.E.L.); (B.M.); (R.M.d.S.); (I.R.)
| | - Renato Martins da Silva
- Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil; (C.L.N.); (A.F.A.); (M.E.L.); (B.M.); (R.M.d.S.); (I.R.)
| | - Katia C. Gondim
- Laboratório de Bioquímica e Fisiologia de Insetos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-617, Brazil; (K.C.G.); (S.S.)
| | - Satoru Konnai
- Laboratory of Infectious Diseases, Hokkaido University, Sapporo 060-0810, Japan;
| | - Isabela Ramos
- Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil; (C.L.N.); (A.F.A.); (M.E.L.); (B.M.); (R.M.d.S.); (I.R.)
| | - Samara Santos
- Laboratório de Bioquímica e Fisiologia de Insetos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-617, Brazil; (K.C.G.); (S.S.)
| | - Alessandra D’Almeida Filardy
- Laboratório de Imunologia Celular, Departamento de Imunologia, Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-617, Brazil; (A.D.F.); (K.G.P.)
| | - Kamila Guimarães Pinto
- Laboratório de Imunologia Celular, Departamento de Imunologia, Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-617, Brazil; (A.D.F.); (K.G.P.)
| | - Itabajara da Silva Vaz Junior
- Centro de Biotecnologia and Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre 91509-900, Brazil;
| | - Carlos Logullo
- Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil; (C.L.N.); (A.F.A.); (M.E.L.); (B.M.); (R.M.d.S.); (I.R.)
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Sriram D, Dayma K, Devi AS, Raghawan AK, Rawat S, Radha V. Complex formation and reciprocal regulation between GSK3β and C3G. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2021; 1868:118964. [PMID: 33450305 DOI: 10.1016/j.bbamcr.2021.118964] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 06/22/2020] [Revised: 01/05/2021] [Accepted: 01/11/2021] [Indexed: 11/16/2022]
Abstract
GSK3β, a ubiquitously expressed Ser/Thr kinase, regulates cell metabolism, proliferation and differentiation. Its activity is spatially and temporally regulated dependent on external stimuli and interacting partners, and its deregulation is associated with various human disorders. In this study, we identify C3G (RapGEF1), a protein essential for mammalian embryonic development as an interacting partner and substrate of GSK3β. In vivo and in vitro interaction assays demonstrated that GSK3β and Akt are present in complex with C3G. Molecular modelling and mutational analysis identified a domain in C3G that aids interaction with GSK3β, and overlaps with its nuclear export sequence. GSK3β phosphorylates C3G on primed as well as unprimed sites, and regulates its subcellular localization. Over-expression of C3G resulted in activation of Akt and inactivation of GSK3β. Huntingtin aggregate formation, dependent on GSK3β inhibition, was enhanced upon C3G overexpression. Stable clones of C2C12 cells generated by CRISPR/Cas9 mediated knockdown of C3G, that cannot differentiate, show reduced Akt activity and S9-GSK3β phosphorylation compared to wild type cells. Co-expression of catalytically active GSK3β inhibited C3G induced myocyte differentiation. C3G mutant defective for GSK3β phosphorylation, does not alter S9-GSK3β phosphorylation and, is compromised for inducing myocyte differentiation. Our results show complex formation and reciprocal regulation between GSK3β and C3G. We have identified a novel function of C3G as a negative regulator of GSK3β, a property important for its ability to induce myogenic differentiation.
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Affiliation(s)
- Divya Sriram
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | - Kunal Dayma
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | - Ambure Sharada Devi
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | | | - Shivali Rawat
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | - Vegesna Radha
- CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
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Structural modeling of GSK3β implicates the inactive (DFG-out) conformation as the target bound by TDZD analogs. Sci Rep 2020; 10:18326. [PMID: 33110096 PMCID: PMC7591898 DOI: 10.1038/s41598-020-75020-w] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Accepted: 09/21/2020] [Indexed: 02/06/2023] Open
Abstract
Glycogen synthase kinase-3β (GSK3β) controls many physiological pathways, and is implicated in many diseases including Alzheimer’s and several cancers. GSK3β-mediated phosphorylation of target residues in microtubule-associated protein tau (MAPTAU) contributes to MAPTAU hyperphosphorylation and subsequent formation of neurofibrillary tangles. Inhibitors of GSK3β protect against Alzheimer’s disease and are therapeutic for several cancers. A thiadiazolidinone drug, TDZD-8, is a non-ATP-competitive inhibitor targeting GSK3β with demonstrated efficacy against multiple diseases. However, no experimental data or models define the binding mode of TDZD-8 with GSK3β, which chiefly reflects our lack of an established inactive conformation for this protein. Here, we used metadynamic simulation to predict the three-dimensional structure of the inactive conformation of GSK3β. Our model predicts that phosphorylation of GSK3β Serine9 would hasten the DFG-flip to an inactive state. Molecular docking and simulation predict the TDZD-8 binding conformation of GSK3β to be inactive, and are consistent with biochemical evidence for the TDZD-8–interacting residues of GSK3β. We also identified the pharmacophore and assessed binding efficacy of second-generation TDZD analogs (TDZD-10 and Tideglusib) that bind GSK3β as non-ATP-competitive inhibitors. Based on these results, the predicted inactive conformation of GSK3β can facilitate the identification of novel GSK3β inhibitors of high potency and specificity.
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Issac PK, Guru A, Chandrakumar SS, Lite C, Saraswathi NT, Arasu MV, Al-Dhabi NA, Arshad A, Arockiaraj J. Molecular process of glucose uptake and glycogen storage due to hamamelitannin via insulin signalling cascade in glucose metabolism. Mol Biol Rep 2020; 47:6727-6740. [PMID: 32809102 DOI: 10.1007/s11033-020-05728-5] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2020] [Accepted: 08/10/2020] [Indexed: 12/19/2022]
Abstract
Understanding the mechanism by which the exogenous biomolecule modulates the GLUT-4 signalling cascade along with the information on glucose metabolism is essential for finding solutions to increasing cases of diabetes and metabolic disease. This study aimed at investigating the effect of hamamelitannin on glycogen synthesis in an insulin resistance model using L6 myotubes. Glucose uptake was determined using 2-deoxy-D-[1-3H] glucose and glycogen synthesis were also estimated in L6 myotubes. The expression levels of key genes and proteins involved in the insulin-signaling pathway were determined using real-time PCR and western blot techniques. The cells treated with various concentrations of hamamelitannin (20 µM to 100 µM) for 24 h showed that, the exposure of hamamelitannin was not cytotoxic to L6 myotubes. Further the 2-deoxy-D-[1-3H] glucose uptake assay was carried out in the presence of wortmannin and Genistein inhibitor for studying the GLUT-4 dependent cell surface recruitment. Hamamelitannin exhibited anti-diabetic activity by displaying a significant increase in glucose uptake (125.1%) and glycogen storage (8.7 mM) in a dose-dependent manner. The optimum concentration evincing maximum activity was found to be 100 µm. In addition, the expression of key genes and proteins involved in the insulin signaling pathway was studied to be upregulated by hamamelitannin treatment. Western blot analysis confirmed the translocation of GLUT-4 protein from an intracellular pool to the plasma membrane. Therefore, it can be conceived that hamamelitannin exhibited an insulinomimetic effect by enhancing the glucose uptake and its further conversion into glycogen by regulating glucose metabolism.
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Affiliation(s)
- Praveen Kumar Issac
- SRM Research Institute, SRM Institute of Science and Technology, Kattankulathur, Chennai, Tamil Nadu, 603 203, India
| | - Ajay Guru
- SRM Research Institute, SRM Institute of Science and Technology, Kattankulathur, Chennai, Tamil Nadu, 603 203, India
| | - Sri Snehaa Chandrakumar
- Department of Biotechnology, Anna University, BIT Campus, Tiruchirappalli, Tamil Nadu, 620 024, India
| | - Christy Lite
- Endocrine and Exposome Laboratory, Department of Zoology, Madras Christian College, Tambaram, Chennai, Tamil Nadu, 600 059, India
| | - N T Saraswathi
- Molecular Biophysics Laboratory, School of Chemical and Biotechnology, SASTRA Deemed to be University, Thanjavur, Tamil Nadu, 613 401, India
| | - Mariadhas Valan Arasu
- Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, 11451, Riyadh, Saudi Arabia
| | - Naif Abdullah Al-Dhabi
- Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, 11451, Riyadh, Saudi Arabia
| | - Aziz Arshad
- International Institute of Aquaculture and Aquatic Sciences (I-AQUAS), Universiti Putra Malaysia, Port Dickson, Negeri Sembilan, 71050, Malaysia
- Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor, 43400, Malaysia
| | - Jesu Arockiaraj
- SRM Research Institute, SRM Institute of Science and Technology, Kattankulathur, Chennai, Tamil Nadu, 603 203, India.
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Huang P, Chen K, Ma T, Cao N, Weng D, Xu C, Xu L. The effects of short-term treatment of microcystin-LR on the insulin pathway in both the HL7702 cell line and livers of mice. ENVIRONMENTAL TOXICOLOGY 2020; 35:727-737. [PMID: 32073747 DOI: 10.1002/tox.22907] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/17/2019] [Revised: 12/07/2019] [Accepted: 01/17/2020] [Indexed: 06/10/2023]
Abstract
Our previous work indicated exposure of Human liver cell 7702 (HL7702) cells to Microcystin-leucine-arginine (MC-LR) for 24 hours can disrupt insulin (INS) signaling by the hyperphosphorylation of specific proteins. For further exploring the time-dependent effect posed by MC-LR on this pathway, in the current study, HL7702 cells together with mice were exposed to the MC-LR with different concentrations under short-term treatment, and then, protein phosphatase 2A (PP2A) activity and expression of proteins related to INS signaling, as well as the characteristics of their action in the liver, were investigated. The results indicated, in HL7702 cells with 0.5, 1, and 6 hours of treatment by MC-LR, PP2A activity showed an obvious decrease in a time and concentration-dependent manner. While the total protein level of Akt, glycogen synthase kinase 3 (GSK-3), and glycogen synthase remained unchanged, GSK-3 and Akt phosphorylation increased significantly. In livers of mice with 1 hour of intraperitoneal injection with MC-LR, a similar change in these proteins was observed. In addition, the levels of total IRS1 and p-IRS1 at serine sites showed decreasing and increasing trends,respectively, and the hematoxylin and eosin staining showed that liver tissues of mice in the maximum-dose group exhibited obvious hepatocyte degeneration and hemorrhage. Our results further proved that short-term treatment with MC-LR can inhibit PP2A activity and disrupt INS signaling proteins' phosphorylation level, thereby interfering with the INS pathway. Our findings provide a helpful understanding of the toxic effects posed by MC-LR on the glucose metabolism of liver via interference with the INS signaling pathway.
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Affiliation(s)
- Pu Huang
- Department of Biochemistry, School of Medicine, Zhejiang University, Hangzhou, China
| | - Kele Chen
- Department of Biochemistry, School of Medicine, Zhejiang University, Hangzhou, China
| | - Tianfeng Ma
- Department I of Clinical Medicine, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
| | - Naifang Cao
- Department I of Clinical Medicine, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
| | - Dengpo Weng
- Department of Biochemistry, School of Medicine, Zhejiang University, Hangzhou, China
| | - Chun Xu
- Department of Endocrinology, The Third Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Lihong Xu
- Department of Biochemistry, School of Medicine, Zhejiang University, Hangzhou, China
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Curcumin attenuates insulin resistance and hepatic lipid accumulation in a rat model of intra-uterine growth restriction through insulin signalling pathway and sterol regulatory element binding proteins. Br J Nutr 2019; 122:616-624. [PMID: 31237229 DOI: 10.1017/s0007114519001508] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The objective of the present study was to investigate the effect of curcumin on insulin resistance (IR) and hepatic lipid accumulation in intra-uterine growth restriction (IUGR). Rats with a normal birth weight (NBW) or IUGR were fed basic diets (NBW and IUGR groups) or basic diets supplemented with curcumin (NBW-C and IUGR-C groups) from 6 to 12 weeks. Rats in the IUGR group showed higher levels of glucose and homeostasis model assessment for insulin resistance index (HOMA-IR) (P < 0·05) than in the NBW group. The livers of IUGR rats exhibited higher (P < 0·05) concentration of TAG and lower (P < 0·05) activities of lipolysis enzymes compared with the normal rats. In response to dietary curcumin supplementation, concentrations of serum insulin, glucose and HOMA-IR, pyruvate, TAG, total cholesterol and NEFA in the liver were decreased (P < 0·05). The concentrations of glycogen and activities of lipolysis enzymes in the liver were increased (P < 0·05) in the IUGR-C group compared with the IUGR group. These results were associated with lower (P < 0·05) phosphorylated insulin receptor substrate 1, protein kinase B or Akt, glycogen synthase kinase 3β and expressions of sterol regulatory element binding protein 1 and fatty acid synthase (FASN); decreased expressions for Cd36, sterol regulatory element binding protein 1c (Srebf1) and Fasn; increased (P < 0·05) expression of PPARα; and expressions for Ppara and hormone-sensitive lipase in the liver of IUGR-C rats than the IUGR rats. Maternal malnutrition caused IR and lipid accumulation in the liver. Curcumin supplementation prevented IR by regulating insulin signalling pathways and attenuated hepatic lipid accumulation.
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Wang L, Zhu Y, Liu X, Chao Z, Wang Y, Zhong T, Guo J, Zhan S, Li L, Zhang H. Glycogen synthase kinase 3β (GSK3β) regulates the expression of MyHC2a in goat skeletal muscle satellite cells (SMSCs). Anim Sci J 2019; 90:1042-1049. [PMID: 31237073 DOI: 10.1111/asj.13253] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 05/12/2019] [Accepted: 05/27/2019] [Indexed: 12/16/2022]
Abstract
Glycogen synthase kinase beta (GSK3β) plays an important role in skeletal muscle growth, regeneration, and repair. However, the mechanism of GSK3β regulating MyHC2a expression is currently not clear. In this study, GSK3β inhibition promoted skeletal muscle satellite cells (SMSCs) differentiation and increased expression of MyoD, MyoG, MyHC1, and MyHC2a genes. Then we cloned approximately 1.1 kb of goat MyHC2a gene promoter. The deletion fragment (-514/+55) of MyHC2a promoter exhibited the highest level of promoter activity, and a NFATc2 element in this region was responsible for MyHC2a promoter activity. Treatment of SB216713 significantly decreased the transcriptional activity of the fragment (-514/+55). Furthermore, GSK3β inhibition had no effect on the luciferase activity of MyHC2a promoter after mutating the NFATc2-binding site. These results demonstrated that GSK3β inhibition promoted SMSCs differentiation and regulated the MyHC2a gene expression through NFATc2 in goat-differentiated SMSCs.
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Affiliation(s)
- Linjie Wang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Yuehua Zhu
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Xin Liu
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Zhe Chao
- Institute of Animal Science and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou, P.R. China
| | - Yan Wang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Tao Zhong
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Jiazhong Guo
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Siyuan Zhan
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Li Li
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Hongping Zhang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
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Transcriptome Changes of Skeletal Muscle RNA-Seq Speculates the Mechanism of Postprandial Hyperglycemia in Diabetic Goto-Kakizaki Rats During the Early Stage of T2D. Genes (Basel) 2019; 10:genes10060406. [PMID: 31141985 PMCID: PMC6627578 DOI: 10.3390/genes10060406] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2019] [Revised: 05/20/2019] [Accepted: 05/23/2019] [Indexed: 12/14/2022] Open
Abstract
To address how skeletal muscle contributes to postprandial hyperglycemia, we performed skeletal muscle transcriptome analysis of diabetic Goto-Kakizaki (GK) and control Wistar rats by RNA sequencing (RNA-Seq). We obtained 600 and 1785 differentially expressed genes in GK rats compared to those Wistar rats at three and four weeks of age, respectively. Specifically, Tbc1d4, involved in glucose uptake, was significantly downregulated in the skeletal muscle of GK aged both three and four weeks compared to those of age-matched Wistar rats. Pdk4, related to glucose uptake and oxidation, was significantly upregulated in the skeletal muscle of GK aged both three and four weeks compared to that of age-matched Wistar rats. Genes (Acadl, Acsl1 and Fabp4) implicated in fatty acid oxidation were significantly upregulated in the skeletal muscle of GK aged four weeks compared to those of age-matched Wistar rats. The overexpression or knockout of Tbc1d4, Pdk4, Acadl, Acsl1 and Fabp4 has been reported to change glucose uptake and fatty acid oxidation directly in rodents. By taking the results of previous studies into consideration, we speculated that dysregulation of key dysregulated genes (Tbc1d4, Pdk4, Acadl, Acsl1 and Fabp4) may lead to a decrease in glucose uptake and oxidation, and an increase in fatty acid oxidation in GK skeletal muscle at three and four weeks, which may, in turn, contribute to postprandial hyperglycemia. Our research revealed transcriptome changes in GK skeletal muscle at three and four weeks. Tbc1d4, Acadl, Acsl1 and Fabp4 were found to be associated with early diabetes in GK rats for the first time, which may provide a new scope for pathogenesis of postprandial hyperglycemia.
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Sarikhani M, Mishra S, Maity S, Kotyada C, Wolfgeher D, Gupta MP, Singh M, Sundaresan NR. SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation. eLife 2018; 7:32952. [PMID: 29504933 PMCID: PMC5860870 DOI: 10.7554/elife.32952] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2017] [Accepted: 03/02/2018] [Indexed: 12/28/2022] Open
Abstract
Glycogen synthase kinase 3 (GSK3) is a critical regulator of diverse cellular functions involved in the maintenance of structure and function. Enzymatic activity of GSK3 is inhibited by N-terminal serine phosphorylation. However, alternate post-translational mechanism(s) responsible for GSK3 inactivation are not characterized. Here, we report that GSK3α and GSK3β are acetylated at Lys246 and Lys183, respectively. Molecular modeling and/or molecular dynamics simulations indicate that acetylation of GSK3 isoforms would hinder both the adenosine binding and prevent stable interactions of the negatively charged phosphates. We found that SIRT2 deacetylates GSK3β, and thus enhances its binding to ATP. Interestingly, the reduced activity of GSK3β is associated with lysine acetylation, but not with phosphorylation at Ser9 in hearts of SIRT2-deficient mice. Moreover, GSK3 is required for the anti-hypertrophic function of SIRT2 in cardiomyocytes. Overall, our study identified lysine acetylation as a novel post-translational modification regulating GSK3 activity.
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Affiliation(s)
- Mohsen Sarikhani
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru, India
| | - Sneha Mishra
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru, India
| | - Sangeeta Maity
- Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru, India
| | - Chaithanya Kotyada
- Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, India
| | - Donald Wolfgeher
- Department of Molecular Genetics and Cell biology, University of Chicago, Chicago, United States
| | - Mahesh P Gupta
- Department of Surgery, University of Chicago, Chicago, United States
| | - Mahavir Singh
- Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, India
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11
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Liu J, Xu C, Zhang S, Li H, Chen K, Huang P, Guo Z, Xu L. Microcystin-LR disrupts insulin signaling by hyperphosphorylating insulin receptor substrate 1 and glycogen synthase. ENVIRONMENTAL TOXICOLOGY 2018; 33:16-22. [PMID: 28984034 DOI: 10.1002/tox.22456] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/14/2017] [Revised: 07/21/2017] [Accepted: 07/27/2017] [Indexed: 06/07/2023]
Abstract
Microcystin-LR (MC-LR) is a cyanobacteria-derived heptapeptide that has been commonly characterized as a hepatotoxin. Although the liver is a primary organ in glucose homeostasis, the effect of MC-LR on glucose metabolism remains unclear. In this study, the human liver cell line HL7702 and ICR mice were exposed to various concentrations of MC-LR for 24 h, and the proteins involved in insulin signaling were investigated. The results showed that MC-LR treatment induced the hyperphosphorylation of insulin receptor substrate 1 (IRS1) at several serine sites, S307, S323, S636/639, and S1101 in HL7702 cells, and S302, S318, S632/635, and S1097 in mice livers. In addition, the activation of S6K1 was demonstrated to play an important role in MC-LR-induced IRS1 hyperphosphorylation at several serine sites. Decreased levels of total IRS1 were observed in the mice livers, but there was no significant change in HL7702 cells. MC-LR also induced glycogen synthase (GS) hyperphosphorylation at S641 (inactivating GS) both in vitro and in vivo, even glycogen synthase kinase 3, a well-known GS kinase, was inactivated after MC-LR treatment. Moreover, MC-LR could block insulin-induced GS activation. In addition, glucose transport in liver cells was not impacted by MC-LR either with or without insulin stimulation. Our study implies that MC-LR can interfere with the actions of IRS1 and GS in insulin signaling and may have a toxic effect on glucose metabolism in the liver.
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Affiliation(s)
- Jinghui Liu
- Department of Biochemistry, School of Medicine, Zhejiang University, Hangzhou, 310058, China
| | - Chun Xu
- Department of Endocrinology, General Hospital of the Chinese People's Armed Police Forces, Beijing, 100039, China
| | - Shaofeng Zhang
- Department of Endocrinology, General Hospital of the Chinese People's Armed Police Forces, Beijing, 100039, China
| | - Haoyan Li
- Department of Endocrinology, General Hospital of the Chinese People's Armed Police Forces, Beijing, 100039, China
| | - Kele Chen
- Department of Biochemistry, School of Medicine, Zhejiang University, Hangzhou, 310058, China
| | - Pu Huang
- Department of Biochemistry, School of Medicine, Zhejiang University, Hangzhou, 310058, China
| | - Zonglou Guo
- Department of Biosystem Engineering, College of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
| | - Lihong Xu
- Department of Biochemistry, School of Medicine, Zhejiang University, Hangzhou, 310058, China
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12
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Wang Y, Wang Y, Zhong T, Guo J, Li L, Zhang H, Wang L. Transcriptional regulation of pig GYS1 gene by glycogen synthase kinase 3β (GSK3β). Mol Cell Biochem 2016; 424:203-208. [PMID: 27785702 DOI: 10.1007/s11010-016-2856-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2016] [Accepted: 10/22/2016] [Indexed: 01/02/2023]
Abstract
Glycogen synthase kinase 3β (GSK3β) is a ubiquitous serine/threonine kinase and has important roles in glycogen metabolism biosynthesis. Studies have revealed that GSK3β can directly regulate the glycogen synthase activity, yet little is known about the regulation of GSK3β on GYS1 gene transcription. Here, we show that overexpression of GSK3β decreased the mRNA expression level of GYS1. Then we cloned approximately 1.5 kb of pig GYS1 gene promoter region, generated sequential deletion constructs, and evaluated their activity. A gradual increase of the promoter activity was seen with increasing length of the promoter sequence, reaching its highest activity to the sequence corresponding to nt -350 to +224, and then decreased. However, the activities of constructed promoter fragments show different responses to GSK3β co-transfection. By analyzing a series of GYS1 promoter reporter constructs, we have defined two crucial regions (-1488 to -539, -350 to -147) that are responsible for GSK3β-induced transcriptional repression. Furthermore, the ChIP results revealed that only the first and second NF-κB sites of GYS1 promoter could bind to p65, and overexpression of GSK3β induced a significant decrease in p65 binding to the second NF-κB binding site, suggesting that GSK3β may regulate expression of GYS1 gene through binding to the second rather than the first NF-κB site. These data suggest that the NF-κB plays important roles in the transcriptional activity of pig GYS1 gene regulated by GSK3β.
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Affiliation(s)
- Yilin Wang
- Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.,Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China
| | - Yan Wang
- Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.,Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China
| | - Tao Zhong
- Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.,Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China
| | - Jiazhong Guo
- Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.,Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China
| | - Li Li
- Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.,Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China
| | - Hongping Zhang
- Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.,Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China
| | - Linjie Wang
- Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China. .,Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
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13
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Abstract
B cell growth and proliferation is tightly regulated by signaling through the B cell receptor and by other membrane bound receptors responding to different cytokines. The PI3K signaling pathway has been shown to play a crucial role in B cell activation, differentiation and survival. Activated B cells undergo metabolic reprograming in response to changing energetic and biosynthetic demands. B cells also need to be able to coordinate metabolic activity and proliferation with nutrient availability. The PI3K signaling network has been implicated in regulating nutrient acquisition, utilization and biosynthesis, thus integrating receptor-mediated signaling with cell metabolism. In this review, we discuss the current knowledge about metabolic changes induced in activated B cells, strategies to adapt to metabolic stress and the role of PI3K signaling in these processes.
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Affiliation(s)
- Julia Jellusova
- a BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg , Freiburg , Germany.,b Max Planck Institute of Immunobiology and Epigenetics , Freiburg , Germany
| | - Robert C Rickert
- c Sanford Burnham Prebys Medical Discovery Institute , La Jolla , CA , USA
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14
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AMPK Facilitates Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550. Mol Cell Biol 2016; 36:1931-42. [PMID: 27161318 DOI: 10.1128/mcb.00118-16] [Citation(s) in RCA: 385] [Impact Index Per Article: 42.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2016] [Accepted: 05/05/2016] [Indexed: 12/12/2022] Open
Abstract
Nrf2 (nuclear factor erythroid 2-related factor 2) is an antioxidant transcription factor. AMP-activated protein kinase (AMPK) functions as a central regulator of cell survival in response to stressful stimuli. Nrf2 should be coordinated with the cell survival pathway controlled by AMPK, but so far the mechanistic connections remain undefined. This study investigated the role of AMPK in Nrf2 trafficking and its activity regulation. A subnetwork integrating neighbor molecules suggested direct interaction between AMPK and Nrf2. In cells, AMPK activation caused nuclear accumulation of Nrf2. In the in vitro kinase and peptide competition assays, AMPK phosphorylated Nrf2 at the Ser558 residue (Ser550 in mouse) located in the canonical nuclear export signal. Nrf2 with an S550A mutation failed to be accumulated in the nucleus after AMPK activation. Leptomycin B, a nuclear export inhibitor, did not enhance nuclear accumulation of wild-type Nrf2 (WT-Nrf2) activated by AMPK or a phospho-Ser550-mimetic Nrf2 mutant, corroborating the finding that AMPK facilitated nuclear accumulation of Nrf2, probably by inhibiting nuclear export. Activated glycogen synthase kinase 3β (GSK3β) diminished the basal nuclear level of Myc-S550A-Nrf2. Taking the data collectively, AMPK phosphorylates Nrf2 at the Ser550 residue, which, in conjunction with AMPK-mediated GSK3β inhibition, promotes nuclear accumulation of Nrf2 for antioxidant response element (ARE)-driven gene transactivation.
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15
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Wang J, Hu S, Jiang W, Song W, Cai L, Wang J. Fucoidan from sea cucumber may improve hepatic inflammatory response and insulin resistance in mice. Int Immunopharmacol 2015; 31:15-23. [PMID: 26690975 DOI: 10.1016/j.intimp.2015.12.009] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2015] [Revised: 12/04/2015] [Accepted: 12/04/2015] [Indexed: 01/13/2023]
Abstract
Nutrition excess-induced inflammation positively contributed to insulin resistance. Fucoidan from sea cucumber can increase glucose translocation in skeletal muscle. However, its effects on inflammation-associated insulin resistance are not understood. We investigated fucoidan from Isostichopus badionotus (Ib-FUC)-alleviated inflammatory response and signaling as well as -improved insulin resistance in the liver of obesity mice. The results showed that Ib-FUC reduced body weight and glucose levels, increased insulin sensitivity, and inhibited serum lipid concentrations. Meanwhile, Hepatic glycogen synthesis was promoted by Ib-FUC via activation of the PI3K/PKB/GSK-3β signaling and regulation of glucose metabolism-related enzymatic activities. Ib-FUC regulated serum inflammatory cytokines and their mRNA expression in the liver. Ib-FUC-induced inactivation of the JNK and IKKβ/NFκB pathways was involved in the activation of insulin signal cascade and inflammatory factor production. These findings suggested that Ib-FUC supplementary-induced alleviation of inflammatory response could be a mechanism responsible for its beneficial effects against hepatic insulin resistance.
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Affiliation(s)
- Jinhui Wang
- Innovation Application Institute, Zhejiang Ocean University, Zhoushan, Zhejiang Province 316022, China
| | - Shiwei Hu
- Innovation Application Institute, Zhejiang Ocean University, Zhoushan, Zhejiang Province 316022, China; College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province 266003, China.
| | - Wei Jiang
- Innovation Application Institute, Zhejiang Ocean University, Zhoushan, Zhejiang Province 316022, China; College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province 266003, China
| | - Wendong Song
- Innovation Application Institute, Zhejiang Ocean University, Zhoushan, Zhejiang Province 316022, China
| | - Lu Cai
- Innovation Application Institute, Zhejiang Ocean University, Zhoushan, Zhejiang Province 316022, China
| | - Jingfeng Wang
- College of Food Science and Engineering, Ocean University of China, Qingdao, Shandong Province 266003, China
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16
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Shen W, Taylor B, Jin Q, Nguyen-Tran V, Meeusen S, Zhang YQ, Kamireddy A, Swafford A, Powers AF, Walker J, Lamb J, Bursalaya B, DiDonato M, Harb G, Qiu M, Filippi CM, Deaton L, Turk CN, Suarez-Pinzon WL, Liu Y, Hao X, Mo T, Yan S, Li J, Herman AE, Hering BJ, Wu T, Martin Seidel H, McNamara P, Glynne R, Laffitte B. Inhibition of DYRK1A and GSK3B induces human β-cell proliferation. Nat Commun 2015; 6:8372. [PMID: 26496802 PMCID: PMC4639830 DOI: 10.1038/ncomms9372] [Citation(s) in RCA: 160] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2015] [Accepted: 08/14/2015] [Indexed: 12/28/2022] Open
Abstract
Insufficient pancreatic β-cell mass or function results in diabetes mellitus. While significant progress has been made in regulating insulin secretion from β-cells in diabetic patients, no pharmacological agents have been described that increase β-cell replication in humans. Here we report aminopyrazine compounds that stimulate robust β-cell proliferation in adult primary islets, most likely as a result of combined inhibition of DYRK1A and GSK3B. Aminopyrazine-treated human islets retain functionality in vitro and after transplantation into diabetic mice. Oral dosing of these compounds in diabetic mice induces β-cell proliferation, increases β-cell mass and insulin content, and improves glycaemic control. Biochemical, genetic and cell biology data point to Dyrk1a as the key molecular target. This study supports the feasibility of treating diabetes with an oral therapy to restore β-cell mass, and highlights a tractable pathway for future drug discovery efforts.
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Affiliation(s)
- Weijun Shen
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Brandon Taylor
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Qihui Jin
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Van Nguyen-Tran
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Shelly Meeusen
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - You-Qing Zhang
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Anwesh Kamireddy
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Austin Swafford
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Andrew F. Powers
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - John Walker
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - John Lamb
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Badry Bursalaya
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Michael DiDonato
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - George Harb
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Minhua Qiu
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Christophe M. Filippi
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Lisa Deaton
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Carolina N. Turk
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Wilma L. Suarez-Pinzon
- Department of Surgery and Schulze Diabetes Institute, University of Minnesota, 420 Delaware Street SE, Minneapolis, Minnesota 55455, USA
| | - Yahu Liu
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Xueshi Hao
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Tingting Mo
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Shanshan Yan
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Jing Li
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Ann E. Herman
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Bernhard J. Hering
- Department of Surgery and Schulze Diabetes Institute, University of Minnesota, 420 Delaware Street SE, Minneapolis, Minnesota 55455, USA
| | - Tom Wu
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - H. Martin Seidel
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Peter McNamara
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Richard Glynne
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
| | - Bryan Laffitte
- Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA
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17
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GSK3-mediated raptor phosphorylation supports amino-acid-dependent mTORC1-directed signalling. Biochem J 2015; 470:207-21. [PMID: 26348909 PMCID: PMC4652938 DOI: 10.1042/bj20150404] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2015] [Accepted: 07/09/2015] [Indexed: 01/06/2023]
Abstract
Glycogen synthase kinase-3 (GSK3) mediates phosphorylation of raptor on Ser859, which crucially supports activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signalling in response to amino acid availability. GSK3 inhibition is associated with reduced mTORC1 signalling that impacts negatively on cell growth, protein synthesis and promotes cellular autophagy. The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser859. We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1 (ribosomal S6 kinase 1) and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation.
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18
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Noel A, Ingrand S, Barrier L. Inhibition of GSK3β by pharmacological modulation of sphingolipid metabolism occurs independently of ganglioside disturbance in a cellular model of Alzheimer's disease. Exp Neurol 2015; 271:308-18. [PMID: 26115843 DOI: 10.1016/j.expneurol.2015.06.021] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2015] [Revised: 06/19/2015] [Accepted: 06/22/2015] [Indexed: 12/11/2022]
Abstract
Accumulating evidence implicates ganglioside and/or related-sphingolipid disturbance in the pathogenesis of Alzheimer's disease (AD). However, it is not known whether these lipidic alterations are connected with other important features of AD, such as deregulated insulin/Akt/GSK3 signaling. In this study, we have treated neuroglioma cells expressing the double Swedish mutation of human amyloid precursor protein (H4APPsw) with several glycosphingolipid (GSL)-modulating agents, and we have analyzed the impact of the aberrant ganglioside composition on the GSK3 activation state. We found that both ceramide analogs D- and L-PDMP (1-phenyl 2-decanoylamino-3-morpholino-1-propanol), which have opposite effects on ganglioside synthesis, selectively inhibited GSK3β via Ser9 phosphorylation independently of the upstream insulin/Akt pathway. Conversely, the iminosugar N-butyldeoxynojirimycin (NB-DNJ) which displayed similar reduction of gangliosides as D-PDMP, did not affect the phosphorylation state of GSK3β. Concurrently, while NB-DNJ did not modify the cellular ceramide content, both PDMP enantiomers strongly and equally reduced the levels of long-chain ceramide species. Altogether, our findings led us to hypothesize that the PDMP-induced altered ganglioside composition is not the principal mechanism involved in the inhibition of GSK3β, but seems to implicate, at least in part, their ability to reduce ceramide levels. Nevertheless, this study provides new information regarding the possibilities to target GSK3β through modulation of sphingolipid metabolism.
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Affiliation(s)
- Anastasia Noel
- Université Laval, Faculté de médecine, Département de Psychiatrie et Neurosciences, Québec, QC, Canada; Centre Hospitalier de l'Université Laval, Axe Neurosciences, 2705 Boulevard Laurier, Québec, QC G1V 4G2, Canada; University of Poitiers, Groupe de Recherche sur le Vieillissement Cérébral, GRéViC EA 3808, Poitiers, France
| | - Sabrina Ingrand
- Université de Poitiers, UFR Médecine & Pharmacie, Service de Biochimie et Toxicologie, 6 rue de la Milétrie, TSA 51115, 86073 Poitiers cedex 9, France
| | - Laurence Barrier
- Université de Poitiers, UFR Médecine & Pharmacie, Service de Biochimie et Toxicologie, 6 rue de la Milétrie, TSA 51115, 86073 Poitiers cedex 9, France.
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19
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González N, Martín-Duce A, Martínez-Arrieta F, Moreno-Villegas Z, Portal-Núñez S, Sanz R, Egido J. Effect of bombesin receptor subtype-3 and its synthetic agonist on signaling, glucose transport and metabolism in myocytes from patients with obesity and type 2 diabetes. Int J Mol Med 2015; 35:925-31. [PMID: 25653074 PMCID: PMC4356436 DOI: 10.3892/ijmm.2015.2090] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2014] [Accepted: 01/15/2015] [Indexed: 11/17/2022] Open
Abstract
Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein-coupled receptor (GPCR) member of the bombesin receptor family. Several studies have suggested an association between obesity, alterations in glucose metabolism, diabetes and the BRS-3 receptor. In this study, we focused on patients simultaneously diagnosed with obesity and type 2 diabetes (OB/T2D). The analysis of BRS-3 expression in the skeletal muscle of these patients revealed a marked decrease in the expression of BRS-3 at the mRNA (23.6±1.3-fold downregulation, p<0.0001) and protein level (49±7% decrease, p<0.05) compared to the normal patients (no obesity and diabetes). Moreover, in cultured primary myocytes from patients with OB/T2D, the synthetic BRS-3 agonist, [D-Try6,β-Ala11,Phe13,Nle14]bombesin6–14, significantly increased the phosphorylation levels of mitogen-activated protein kinase (MAPK), p90RSK1, protein kinase B (PKB) and p70s6K. Specifically, the ligand at 10−11 M induced the maximal phosphorylation of MAPKs (p42, 159±15% of the control; p44, 166±11% of the control; p<0.0001) and p90RSK1 (148±2% of the control, p<0.0001). The basal phosphorylation levels of all kinases were reduced (p<0.05) in the patients with OB/T2D compared to the normal patients. Furthermore, the BRS-3 agonist stimulated glucose transport, which was already detected at 10−12 M (133±9% of the control), reached maximal levels at 10−11 M (160±9%, p<0.0001) and was maintained at up to 10−8 M (overall mean, 153±7%; p<0.007). This effect was less promiment than that attained with 10−8 M insulin (202±9%, p=0.009). The effect of the agonist on glycogen synthase a activity achieved the maximum effect at 10−11 M (165±16% of the control; p<0.0001), which did not differ from that observed with higher concentrations of the agonist. These results suggest that muscle cells isolated from patients with OB/T2D have extremely high sensitivity to the synthetic ligand, and the effects are particularly observed on MAPK and p90RSK1 phosphorylation, as well as glucose uptake. Moreover, our data indicate that BRS-3 may prove to be useful as a potential therapeutic target for the treatment of patients with OB/T2D.
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Affiliation(s)
- Nieves González
- Renal, Vascular and Diabetes Research Laboratory, IIS-Jiménez Díaz Foundation, The Autonomous University of Madrid, Spanish Biomedical Research Network in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain
| | | | - Félix Martínez-Arrieta
- Department of General Surgery, Puerta de Hierro-Majadahonda University Hospital, The Autonomous University of Madrid, Madrid, Spain
| | - Zaida Moreno-Villegas
- Renal, Vascular and Diabetes Research Laboratory, IIS-Jiménez Díaz Foundation, The Autonomous University of Madrid, Madrid, Spain
| | - Sergio Portal-Núñez
- Department of Bone and Mineral Metabolism, IIS-Jiménez Díaz Foundation, Cooperative Research Thematic Network on Aging and Frailty (RETICEF), Madrid, Spain
| | - Raúl Sanz
- Department of Neurology, IIS-Jiménez Díaz Foundation, Madrid, Spain
| | - Jesús Egido
- Renal, Vascular and Diabetes Research Laboratory, IIS-Jiménez Díaz Foundation, The Autonomous University of Madrid, Spanish Biomedical Research Network in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain
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20
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Song WJ, Song EAC, Jung MS, Choi SH, Baik HH, Jin BK, Kim JH, Chung SH. Phosphorylation and inactivation of glycogen synthase kinase 3β (GSK3β) by dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A). J Biol Chem 2015; 290:2321-33. [PMID: 25477508 PMCID: PMC4303684 DOI: 10.1074/jbc.m114.594952] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2014] [Revised: 11/14/2014] [Indexed: 12/27/2022] Open
Abstract
Glycogen synthase kinase 3β (GSK3β) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3β by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3β is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3β directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr(356) residue inhibits GSK3β activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3β on obesity. This result suggests a potential in vivo association between GSK3β and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)(356)-GSK3β was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3β activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3β activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3β.
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Affiliation(s)
- Woo-Joo Song
- From the Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, School of Medicine, the Institute for Brain Science and Technology, Inje University, Busan 614-735, Korea
| | - Eun-Ah Christine Song
- From the Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, School of Medicine, the Institute for Brain Science and Technology, Inje University, Busan 614-735, Korea
| | - Min-Su Jung
- the Institute for Brain Science and Technology, Inje University, Busan 614-735, Korea
| | - Sun-Hee Choi
- the Institute for Brain Science and Technology, Inje University, Busan 614-735, Korea
| | - Hyung-Hwan Baik
- From the Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, School of Medicine
| | - Byung Kwan Jin
- From the Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, School of Medicine
| | - Jeong Hee Kim
- Department of Oral Biochemistry and Molecular Biology, School of Dentistry, and Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Korea and
| | - Sul-Hee Chung
- From the Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, School of Medicine, the Institute for Brain Science and Technology, Inje University, Busan 614-735, Korea
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Over-expression of muscle glycogen synthase in human diabetic nephropathy. Histochem Cell Biol 2014; 143:313-24. [PMID: 25371328 DOI: 10.1007/s00418-014-1290-2] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/23/2014] [Indexed: 01/15/2023]
Abstract
Diabetic nephropathy (DN) is a major complication of diabetic patients and the leading cause of end-stage renal disease. Glomerular dysfunction plays a critical role in DN, but deterioration of renal function also correlates with tubular alterations. Human DN is characterized by glycogen accumulation in tubules. Although this pathological feature has long been recognized, little information exists about the triggering mechanism. In this study, we detected over-expression of muscle glycogen synthase (MGS) in diabetic human kidney. This enhanced expression suggests the participation of MGS in renal metabolic changes associated with diabetes. HK2 human renal cell line exhibited an intrinsic ability to synthesize glycogen, which was enhanced after over-expression of protein targeting to glycogen. A correlation between increased glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogen-induced damage during long-term diabetes.
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Sue YM, Chou HC, Chang CC, Yang NJ, Chou Y, Juan SH. L-carnitine protects against carboplatin-mediated renal injury: AMPK- and PPARα-dependent inactivation of NFAT3. PLoS One 2014; 9:e104079. [PMID: 25090113 PMCID: PMC4121315 DOI: 10.1371/journal.pone.0104079] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2014] [Accepted: 07/06/2014] [Indexed: 12/30/2022] Open
Abstract
We have previously shown that carboplatin induces inflammation and apoptosis in renal tubular cells (RTCs) through the activation of the nuclear factor of activated T cells-3 (NFAT3) protein by reactive oxygen species (ROS), and that the ROS-mediated activation of NFAT3 is prevented by N-acetyl cysteine and heme oxygenase-1 treatment. In the current study, we investigated the underlying molecular mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Balb/c mice and RTCs were used as model systems. Carboplatin-induced apoptosis in RTCs was examined using terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling. We evaluated the effects of the overexpression of the peroxisome-proliferator-activated receptor alpha (PPARα) protein, the knockdown of PPARα gene, and the blockade of AMPK activation and PPARα to investigate the underlying mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Carboplatin reduced the nuclear translocation, phosphorylation, and peroxisome proliferator responsive element transactivational activity of PPARα. These carboplatin-mediated effects were prevented by L-carnitine through a mechanism dependent on AMPK phosphorylation and subsequent PPARα activation. The activation of PPARα induced cyclooxygenase 2 (COX-2) and prostacyclin (PGI2) synthase expression that formed a positive feedback loop to further activate PPARα. The coimmunoprecipitation of the nuclear factor (NF) κB proteins increased following the induction of PPARα by L-carnitine, which reduced NFκB transactivational activity and cytokine expression. The in vivo study showed that the inactivation of AMPK suppressed the protective effect of L-carnitine in carboplatin-treated mice, indicating that AMPK phosphorylation is required for PPARα activation in the L-carnitine-mediated protection of RTC apoptosis caused by carboplatin. The results of our study provide molecular evidence that L-carnitine prevents carboplatin-mediated apoptosis through AMPK-mediated PPARα activation.
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Affiliation(s)
- Yuh-Mou Sue
- Department of Nephrology, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan
| | - Hsiu-Chu Chou
- Department of Anatomy, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Chih-Cheng Chang
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
- Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Nian-Jie Yang
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
- Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Ying Chou
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
- Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Shu-Hui Juan
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
- Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
- * E-mail:
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Ramos-Álvarez I, Moreno-Villegas Z, Martín-Duce A, Sanz R, Aparicio C, Portal-Núñez S, Mantey SA, Jensen RT, González N. Human BRS-3 receptor: functions/role in cell signaling pathways and glucose metabolism in obese or diabetic myocytes. Peptides 2014; 51:91-99. [PMID: 24220502 DOI: 10.1016/j.peptides.2013.11.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/16/2013] [Revised: 11/04/2013] [Accepted: 11/04/2013] [Indexed: 10/26/2022]
Abstract
Several studies showed that the orphan Bombesin Receptor Subtype-3 (BRS-3) - member of the bombesin receptor family - has an important role in glucose homeostasis (v.g.: BRS-3-KO mice developed mild obesity, and decreased levels of BRS-3 mRNA/protein have been described in muscle from obese (OB) and type 2 diabetic (T2D) patients). In this work, to gain insight into BRS-3 receptor cell signaling pathways, and its implication on glucose metabolism, primary cultured myocytes from normal subjects, OB or T2D patients were tested using high affinity ligand - [d-Tyr(6),β-Ala(11),Phe(13),Nle(14)]bombesin6-14. In muscle cells from all metabolic conditions, the compound significantly increased not only MAPKs, p90RSK1, PKB and p70s6K phosphorylation levels, but also PI3K activity; moreover, it produced a dose-response stimulation of glycogen synthase a activity and glycogen synthesis. Myocytes from OB and T2D patients were more sensitive to the ligand than normal, and T2D cells even more than obese myocytes. These results widen the knowledge of human BRS-3 cell signaling pathways induced by a BRS-3 agonist, described its insulin-mimetic effects on glucose metabolism, showed the role of BRS-3 receptor in glucose homeostasis, and also propose the employing of BRS-3/ligand system, as participant in the obese and diabetic therapies.
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MESH Headings
- Adult
- Aged
- Bombesin/pharmacology
- Cells, Cultured
- Diabetes Mellitus, Type 2/metabolism
- Diabetes Mellitus, Type 2/pathology
- Female
- Glucose/metabolism
- Glycogen/biosynthesis
- Glycogen Synthase/metabolism
- Homeostasis
- Humans
- Male
- Middle Aged
- Mitogen-Activated Protein Kinases/metabolism
- Muscle Fibers, Skeletal/drug effects
- Muscle Fibers, Skeletal/metabolism
- Obesity/metabolism
- Obesity/pathology
- Peptide Fragments/pharmacology
- Phosphatidylinositol 3-Kinases/metabolism
- Phosphorylation
- Protein Processing, Post-Translational
- Proto-Oncogene Proteins c-akt/metabolism
- Receptors, Bombesin/agonists
- Receptors, Bombesin/physiology
- Ribosomal Protein S6 Kinases, 70-kDa/metabolism
- Ribosomal Protein S6 Kinases, 90-kDa/metabolism
- Signal Transduction
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Affiliation(s)
- I Ramos-Álvarez
- Department of Metabolism, Nutrition and Hormones, IIS-Fundación Jiménez Díaz, CIBERDEM, Madrid, Spain
| | - Z Moreno-Villegas
- Department of Metabolism, Nutrition and Hormones, IIS-Fundación Jiménez Díaz, CIBERDEM, Madrid, Spain
| | - A Martín-Duce
- Department of Nursery, Unit of Surgery, Universidad de Alcalá de Henares, Madrid, Spain
| | - R Sanz
- Department of Neurology, IIS-Fundación Jiménez Díaz, Madrid, Spain
| | - C Aparicio
- Department of Vascular Surgery, Fundación Jiménez Díaz, Madrid, Spain
| | - S Portal-Núñez
- Department of Bone Mineral Metabolism, IIS-Fundación Jiménez Díaz, Madrid, Spain
| | - S A Mantey
- Digestive Diseases Branch, NIDDK, NIH, Bethesda, USA
| | - R T Jensen
- Digestive Diseases Branch, NIDDK, NIH, Bethesda, USA
| | - N González
- Department of Metabolism, Nutrition and Hormones, IIS-Fundación Jiménez Díaz, CIBERDEM, Madrid, Spain.
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ElAli A, Thériault P, Préfontaine P, Rivest S. Mild chronic cerebral hypoperfusion induces neurovascular dysfunction, triggering peripheral beta-amyloid brain entry and aggregation. Acta Neuropathol Commun 2013; 1:75. [PMID: 24252187 PMCID: PMC3843528 DOI: 10.1186/2051-5960-1-75] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2013] [Accepted: 11/06/2013] [Indexed: 11/23/2022] Open
Abstract
Background The Blood–brain barrier (BBB) controls brain supply with oxygen and nutrients, and protects the brain from toxic metabolites, such as beta-amyloid (Aβ) peptides. The neurovascular unit (NVU) couples vascular and neuronal functions by controlling BBB parameters based on brain needs. As such, NVU/BBB dysfunction, associated to irregularities in cerebral blood flow (CBF), has been proposed to contribute in the pathogenesis of Alzheimer’s disease (AD), mainly by impairing cerebral Aβ clearance. However, the spatiotemporal contribution of the NVU/BBB in the neurodegenerative cascades remains elusive. Results By using C57BL/6J mice subjected to right common carotid artery (rCCA) permanent ligation in order to induce mild chronic cerebral hypoperfusion, we show here that cerebral hypoperfusion induced NVU dysfunction by reducing ABCB1 protein expression in brain capillaries. ABCB1 reduction was mainly triggered by an enhanced Glycogen Synthase Kinase 3 (GSK3β) activation, which decreased β-catenin nuclear abundance. Moreover, cerebral hypoperfusion triggered early vascular deposition of peripherally applied human Aβ1-42 peptides, which has shifted from highly vascular to the parenchyma 6 weeks later, forming small stable Aβ deposits. Hypoperfusion induced a deregulation in glucose metabolism, as brain reperfusion, or the administration of a high dose of glucose, diminished GSK3β activation, recuperated β-catenin nuclear abundance, reestablished ABCB1 protein expression, and prevented Aβ vascular early deposition. These results demonstrate that mild chronic cerebral hypoperfusion creates a metabolically deregulated microenvironment, thus triggering the brain entry and aggregation of peripherally applied human Aβ1-42 peptides. Conclusion Our study offers new insights on the initiation of the neurodegenerative cascades observed in AD, which could be valuable in developing adequate treatment strategies.
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Flepisi TB, Lochner A, Huisamen B. The Consequences of Long-Term Glycogen Synthase Kinase-3 Inhibition on Normal and Insulin Resistant Rat Hearts. Cardiovasc Drugs Ther 2013; 27:381-92. [DOI: 10.1007/s10557-013-6467-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
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Kaneki M, Fukushima Y, Shinozaki S, Fukaya M, Habiro M, Shimizu N, Chang K, Yasuhara S, Martyn JAJ. iNOS inhibitor, L-NIL, reverses burn-induced glycogen synthase kinase-3β activation in skeletal muscle of rats. Metabolism 2013; 62:341-6. [PMID: 22995863 PMCID: PMC4090935 DOI: 10.1016/j.metabol.2012.08.010] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/21/2012] [Revised: 08/16/2012] [Accepted: 08/16/2012] [Indexed: 11/23/2022]
Abstract
OBJECTIVES Recent studies suggest that activation of glycogen synthase kinase (GSK)-3β may be involved in burn injury-induced metabolic derangements and protein breakdown in skeletal muscle. However, the mechanism for GSK-3β activation after burn injury is unknown. To investigate the role of inducible nitric oxide synthase (iNOS) in this scenario, a major mediator of inflammation, we examined the effects of a specific inhibitor for iNOS, L-NIL, on GSK-3β activity in skeletal muscle of burned rats. MATERIALS/METHODS Full-thickness third degree burn injury comprising 40% of total body surface area was produced under anesthesia in male Sprague-Dawley rats (160-190g) by immersing the back of the trunk for 15s and the abdomen for 8s in 80°C water. Burned and sham-burned rats were treated with L-NIL (60mg/kg BW, b.i.d., IP) or phosphate-buffered saline for three days. GSK-3β activity in skeletal muscle was evaluated by immune complex kinase assay, and by phosphorylation status of GSK-3β and its endogenous substrate, glycogen synthase. RESULTS GSK-3β activity was increased in a time-dependent manner in skeletal muscle after burn injury, concomitant with the induction of iNOS expression. iNOS inhibitor, L-NIL, reverted the elevated GSK-3β activity in skeletal muscle of burned rats, although L-NIL did not alter GSK-3β activity in sham-burned rats. CONCLUSIONS Our results clearly indicate that iNOS plays an important role in burn injury-induced GSK-3β activation in skeletal muscle. These findings suggest that iNOS may contribute to burn injury-induced metabolic derangements, in part, by activating GSK-3β.
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Affiliation(s)
- Masao Kaneki
- Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, Boston, MA 02114, USA.
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27
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Montori-Grau M, Tarrats N, Osorio-Conles O, Orozco A, Serrano-Marco L, Vázquez-Carrera M, Gómez-Foix AM. Glucose dependence of glycogen synthase activity regulation by GSK3 and MEK/ERK inhibitors and angiotensin-(1-7) action on these pathways in cultured human myotubes. Cell Signal 2013; 25:1318-27. [PMID: 23453973 DOI: 10.1016/j.cellsig.2013.02.014] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2012] [Revised: 01/31/2013] [Accepted: 02/13/2013] [Indexed: 11/18/2022]
Abstract
Glycogen synthase (GS) is activated by glucose/glycogen depletion in skeletal muscle cells, but the contributing signaling pathways, including the chief GS regulator GSK3, have not been fully defined. The MEK/ERK pathway is known to regulate GSK3 and respond to glucose. The aim of this study was to elucidate the GSK3 and MEK/ERK pathway contribution to GS activation by glucose deprivation in cultured human myotubes. Moreover, we tested the glucose-dependence of GSK3 and MEK/ERK effects on GS and angiotensin (1-7) actions on these pathways. We show that glucose deprivation activated GS, but did not change phospho-GS (Ser640/1), GSK3β activity or activity-activating phosphorylation of ERK1/2. We then treated glucose-replete and -depleted cells with SB415286, U0126, LY294 and rapamycin to inhibit GSK3, MEK1/2, PI3K and mTOR, respectively. SB415286 activated GS and decreased the relative phospho-GS (Ser640/1) level, more in glucose-depleted than -replete cells. U0126 activated GS and reduced the phospho-GS (Ser640/1) content significantly in glucose-depleted cells, while GSK3β activity tended to increase. LY294 inactivated GS in glucose-depleted cells only, without affecting relative phospho-GS (Ser640/1) level. Rapamycin had no effect on GS activation. Angiotensin-(1-7) raised phospho-ERK1/2 but not phospho-GSK3β (Ser9) content, while it inactivated GS and increased GS phosphorylation on Ser640/1, in glucose-replete cells. In glucose-depleted cells, angiotensin-(1-7) effects on ERK1/2 and GS were reverted, while relative phospho-GSK3β (Ser9) content decreased. In conclusion, activation of GS by glucose deprivation is not due to GS Ser640/1 dephosphorylation, GSK3β or ERK1/2 regulation in cultured myotubes. However, glucose depletion enhances GS activation/Ser640/1 dephosphorylation due to both GSK3 and MEK/ERK inhibition. Angiotensin-(1-7) inactivates GS in glucose-replete cells in association with ERK1/2 activation, not with GSK3 regulation, and glucose deprivation reverts both hormone effects. Thus, the ERK1/2 pathway negatively regulates GS activity in myotubes, without involving GSK3 regulation, and as a function of the presence of glucose.
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Affiliation(s)
- Marta Montori-Grau
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM)-Instituto de Salud Carlos III, Spain.
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Lee S, Yang WK, Song JH, Ra YM, Jeong JH, Choe W, Kang I, Kim SS, Ha J. Anti-obesity effects of 3-hydroxychromone derivative, a novel small-molecule inhibitor of glycogen synthase kinase-3. Biochem Pharmacol 2013; 85:965-76. [PMID: 23337568 DOI: 10.1016/j.bcp.2012.12.023] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2012] [Revised: 12/27/2012] [Accepted: 12/27/2012] [Indexed: 11/30/2022]
Abstract
Glycogen synthase kinase 3 (GSK-3) plays a central role in cellular energy metabolism, and dysregulation of GSK-3 activity is implicated in a variety of metabolic disorders, including obesity, type 2 diabetes, and cancer. Hence, GSK-3 has emerged as an attractive target molecule for the treatment of metabolic disorders. Therefore, this research focused on identification and characterization of a novel small-molecule GSK-3 inhibitor. Compound 1a, a structure based on 3-hydroxychromone bearing isothiazolidine-1,1-dione, was identified from chemical library as a highly potent GSK-3 inhibitor. An in vitro kinase assay utilizing a panel of kinases demonstrated that compound 1a strongly inhibits GSK-3β. The potential effects of compound 1a on the inactivation of GSK-3 were confirmed in human liver HepG2 and human embryonic kidney HEK293 cells. Stabilization of glycogen synthase and β-catenin, which are direct targets of GSK-3, by compound 1a was assessed in comparison with two other GSK-3 inhibitors: LiCl and SB-415286. In mouse 3T3-L1 preadipocytes, compound 1a markedly blocked adipocyte differentiation. Consistently, intraperitoneal administration of compound 1a to diet-induced obese mice significantly ameliorated their key symptoms such as body weight gain, increased adiposity, dyslipidemia, and hepatic steatosis due to the marked reduction of whole-body lipid level. In vitro and in vivo effects were accompanied by upregulation of β-catenin stability and downregulation of the expression of several critical genes related to lipid metabolism. From these results, it can be concluded that compound 1a, a novel small-molecule inhibitor of GSK-3, has potential as a new class of therapeutic agent for obesity treatment.
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Affiliation(s)
- Sooho Lee
- Department of Biochemistry and Molecular Biology, Medical Research Center and Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea
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Wang L, Zuo B, Xu D, Ren Z, Zhang H, Li X, Lei M, Xiong Y. Alternative splicing of the porcine glycogen synthase kinase 3β (GSK-3β) gene with differential expression patterns and regulatory functions. PLoS One 2012; 7:e40250. [PMID: 22792253 PMCID: PMC3391277 DOI: 10.1371/journal.pone.0040250] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2011] [Accepted: 06/04/2012] [Indexed: 01/07/2023] Open
Abstract
Background Glycogen synthase kinase 3 (GSK3α and GSK3β) are serine/threonine kinases involved in numerous cellular processes and diverse diseases including mood disorders, Alzheimer’s disease, diabetes, and cancer. However, in pigs, the information on GSK3 is very limited. Identification and characterization of pig GSK3 are not only important for pig genetic improvement, but also contribute to the understanding and development of porcine models for human disease prevention and treatment. Methodology Five different isoforms of GSK3β were identified in porcine different tissues, in which three isoforms are novel. These isoforms had differential expression patterns in the fetal and adult of the porcine different tissues. The mRNA expression level of GSK3β isoforms was differentially regulated during the course of the insulin treatment, suggesting that different GSK3β isoforms may have different roles in insulin signaling pathway. Moreover, GSK3β5 had a different role on regulating the glycogen synthase activity, phosphorylation and the expression of porcine GYS1 and GYS2 gene compared to other GSK3β isoforms. Conclusions We are the first to report five different isoforms of GSK3β identified from the porcine different tissues. Splice variants of GSK3β exhibit differential activity towards glycogen synthase. These results provide new insight into roles of the GSK3β on regulating glycogen metabolism.
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Affiliation(s)
- Linjie Wang
- Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Ya’an, Sichuan, People’s Republic of China
- * E-mail: (LW); (YX)
| | - Bo Zuo
- Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture & Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, People’s Republic of China
| | - Dequan Xu
- Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture & Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, People’s Republic of China
| | - Zuqing Ren
- Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture & Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, People’s Republic of China
| | - Hongping Zhang
- Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Ya’an, Sichuan, People’s Republic of China
| | - Xuewei Li
- Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Ya’an, Sichuan, People’s Republic of China
| | - Minggang Lei
- Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture & Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, People’s Republic of China
| | - Yuanzhu Xiong
- Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture & Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, People’s Republic of China
- * E-mail: (LW); (YX)
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Abstract
Acute myeloid leukemia (AML) is the most common form of leukemia in adults. Unfortunately, the standard therapeutic agents used for this disease have high toxicities and poor efficacy. The one exception to these poor outcomes is the use of the retinoid, all-trans retinoic acid (ATRA), for a rare subtype of AML (APL). The use of the differentiation agent, ATRA, in combination with low-dose chemotherapy leads to the long-term survival and presumed cure of 75-85% of patients. Unfortunately ATRA has not been clinically useful for other subtypes of AML. Though many non-APL leukemic cells respond to ATRA, they require significantly higher concentrations of ATRA for effective differentiation. Here we show that the combination of ATRA with glycogen synthase kinase 3 (GSK3) inhibition significantly enhances ATRA-mediated AML differentiation and growth inhibition. These studies have revealed that ATRA's receptor, the retinoic acid receptor (RAR), is a novel target of GSK3 phosphorylation and that GSK3 can impact the expression and transcriptional activity of the RAR. Overall, our studies suggest the clinical potential of ATRA and GSK3 inhibition for AML and provide a mechanistic framework to explain the promising activity of this combination regimen.
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Wang ZB, Zeng HC, Wei HS, Yi GH, Yu J, Wang YT, Zhang YL, Yin WD. NO-1886 ameliorates glycogen metabolism in insulin-resistant HepG2 cells by GSK-3β signalling. ACTA ACUST UNITED AC 2011; 64:293-301. [PMID: 22221106 DOI: 10.1111/j.2042-7158.2011.01402.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
OBJECTIVES The aim of the study was to elucidate the possible role and mechanism of NO-1886 (ibrolipim, a lipoprotein lipase activator) in ameliorating insulin resistance induced by high palmitate. METHODS HepG2 cells were cultured in RPMI 1640 medium and were treated with palmitate to induce insulin resistance. Free fatty acids (FFAs), glucose, glycogen, cell viability and mRNA and protein levels were analysed separately. KEY FINDINGS We found that HepG2 cells treated with 0.5 mm palmitate for 48 h led to a significant decrease of insulin-induced glucose consumption (from 2.89 ± 0.85 mm in the control to 0.57 ± 0.44 mm in palmitate). Insulin resistance (IR) of HepG2 cells was induced by 0.5 mm palmitate for 48 h. NO-1886 stimulated glucose consumption, glycogen synthesis and FFA absorption in insulin-resistant HepG2 cells. Maximum stimulation effects were observed with 10 µm NO-1886 for 24 h. Compared with the dimethyl sulfoxide-treated group, 2.5 µm NO-1886 or higher could induce the mRNA expression of lipoprotein lipase. Meanwhile, NO-1886 increased the protein content of P-GSK-3βser(9) and decreased the protein level of GSK-3β in insulin-resistant HepG2 cells, but NO-1886 didn't change the protein levels of PI3-Kp85 and Akt2. CONCLUSION Lipoprotein lipase activator NO-1886 could increase glycogen synthesis in HepG2 cells and could ameliorate the insulin resistance, which was associated with GSK-3 signalling.
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Affiliation(s)
- Zong-Bao Wang
- Key Laboratory for Arteriosclerology of Hunan Province, University of South China, Hengyang, Hunan, China
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Wang L, Xiong Y, Zuo B, Lei M, Ren Z, Xu D. Molecular and functional characterization of glycogen synthase in the porcine satellite cells under insulin treatment. Mol Cell Biochem 2011; 360:169-80. [PMID: 21931959 DOI: 10.1007/s11010-011-1054-4] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2011] [Accepted: 09/08/2011] [Indexed: 12/24/2022]
Abstract
Glycogen synthase (GS) catalyzes the key step of glycogen synthesis and plays an important role in glycogen metabolism in liver and muscle. In this study, we cloned the cDNA and promoter sequences of porcine glycogen synthesis genes (GYS1 and GYS2). Expression analysis revealed that porcine GYS1 was highly expressed in the skeletal muscle and heart. GYS2 was expressed specifically in liver and subcutaneous adipose tissue. The expression level of GYS1 was up-regulated from proliferation to differentiation in the porcine satellite cells, and insulin did not significantly affect the transcription of GYS1. Insulin stimulated 72-h-differentiated satellite cells as indicated by decrease in phosphorylation of GS, but did not affect GYS1 transcription and total GS protein level, suggesting that the effect of insulin is primarily mediated via posttranscriptional control rather than regulated at the transcriptional level. Four single-nucleotide polymorphisms (SNPs) were detected in the promoter and cDNA sequences of porcine GYS1. Association analyses revealed that the GYS1 Hin6I and MvaI polymorphisms both had significant associations (P < 0.05) with pH of M. longissimus dorsi (pHLD), M. biceps femoris (pHBF) and M. semipinalis capitis (pHSC) at 45 min postmortem. These results provide useful information for further investigation on the function of glycogen synthase in porcine skeletal muscle.
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Affiliation(s)
- Linjie Wang
- Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China
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Hutchinson DS, Catus SL, Merlin J, Summers RJ, Gibbs ME. α₂-Adrenoceptors activate noradrenaline-mediated glycogen turnover in chick astrocytes. J Neurochem 2011; 117:915-26. [PMID: 21447002 DOI: 10.1111/j.1471-4159.2011.07261.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
In the brain, glycogen is primarily stored in astrocytes where it is regulated by several hormones/neurotransmitters, including noradrenaline that controls glycogen breakdown (in the short term) and synthesis. Here, we have examined the adrenoceptor (AR) subtype that mediates the glycogenic effect of noradrenaline in chick primary astrocytes by the measurement of glycogen turnover (total (14) C incorporation of glucose into glycogen) following noradrenergic activation. Noradrenaline and insulin increased glycogen turnover in a concentration-dependent manner. The effect of noradrenaline was mimicked by stimulation of α(2) -ARs (and to a lesser degree by β(3) -ARs), but not by stimulation of α(1) -, β(1) -, or β(2) -ARs, and occurred only in astrocytes and not neurons. In chick astrocytes, studies using RT-PCR and radioligand binding showed that α(2A) - and α(2C) -AR mRNA and protein were present. α(2) -AR- or insulin-mediated glycogen turnover was inhibited by phosphatidylinositol-3 kinase inhibitors, and both insulin and clonidine caused phosphorylation of Akt and glycogen synthase kinase-3 in chick astrocytes. α(2) -AR but not insulin-mediated glycogen turnover was inhibited by pertussis toxin pre-treatment indicating involvement of Gi/o proteins. These results show that the increase in glycogen turnover caused by noradrenaline is because of activation of α(2) -ARs that increase glycogen turnover in astrocytes utilizing a Gi/o-PI3K pathway.
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Affiliation(s)
- Dana S Hutchinson
- Department of Pharmacology, Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, Victoria 3052, Australia.
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Wei M, Wang Z, Yao H, Yang Z, Zhang Q, Liu B, Yu Y, Su L, Zhu Z, Gu Q. P27(Kip1), regulated by glycogen synthase kinase-3β, results in HMBA-induced differentiation of human gastric cancer cells. BMC Cancer 2011; 11:109. [PMID: 21439087 PMCID: PMC3078896 DOI: 10.1186/1471-2407-11-109] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2011] [Accepted: 03/27/2011] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Gastric cancer is the second most common cause of global cancer-related mortality. Although dedifferentiation predicts poor prognosis in gastric cancer, the molecular mechanism underlying dedifferentiation, which could provide fundamental insights into tumor development and progression, has yet to be elucidated. Furthermore, the molecular mechanism underlying the effects of hexamethylene bisacetamide (HMBA), a recently discovered differentiation inducer, requires investigation and there are no reported studies concerning the effect of HMBA on gastric cancer. METHODS Based on the results of FACS analysis, the levels of proteins involved in the cell cycle or apoptosis were determined using western blotting after single treatments and sequential combinations of HMBA and LiCl. GSK-3β and proton pump were investigated by western blotting after up-regulating Akt expression by Ad-Akt infection. To investigate the effects of HMBA on protein localization and the activities of GSK-3β, CDK2 and CDK4, kinase assays, immunoprecipitation and western blotting were performed. In addition, northern blotting and RNase protection assays were carried out to determine the functional concentration of HMBA. RESULTS HMBA increased p27(Kip1) expression and induced cell cycle arrest associated with gastric epithelial cell differentiation. In addition, treating gastric-derived cells with HMBA induced G0/G1 arrest and up-regulation of the proton pump, a marker of gastric cancer differentiation. Moreover, treatment with HMBA increased the expression and activity of GSK-3β in the nucleus but not the cytosol. HMBA decreased CDK2 activity and induced p27(Kip1) expression, which could be rescued by inhibition of GSK-3β. Furthermore, HMBA increased p27(Kip1) binding to CDK2, and this was abolished by GSK-3β inhibition. CONCLUSIONS The results presented herein suggest that GSK-3β functions by regulating p27(Kip1) assembly with CDK2, thereby playing a critical role in G0/G1 arrest associated with HMBA-induced gastric epithelial cell differentiation.
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Affiliation(s)
- Min Wei
- Key Laboratory of Shanghai Gastric Neoplasms, Department of Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
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Raben N, Schreiner C, Baum R, Takikita S, Xu S, Xie T, Myerowitz R, Komatsu M, Van der Meulen JH, Nagaraju K, Ralston E, Plotz PH. Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease. Autophagy 2011; 6:1078-89. [PMID: 20861693 DOI: 10.4161/auto.6.8.13378] [Citation(s) in RCA: 128] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alphaglucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.
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Affiliation(s)
- Nina Raben
- Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA.
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Roe CR, Bottiglieri T, Wallace M, Arning E, Martin A. Adult Polyglucosan Body Disease (APBD): Anaplerotic diet therapy (Triheptanoin) and demonstration of defective methylation pathways. Mol Genet Metab 2010; 101:246-52. [PMID: 20655781 DOI: 10.1016/j.ymgme.2010.06.017] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/03/2010] [Accepted: 06/03/2010] [Indexed: 10/19/2022]
Abstract
APBD is a rare disorder most often affecting adults of Ashkenazi Jewish origin due to partial deficiency of the glycogen brancher enzyme (GBE). It is characterized by progressive involvement of both the central and peripheral nervous systems and deposition of amylopectin-like polyglucosan bodies. There have been no metabolic derangements that might suggest effective therapy nor have there been any clinical improvements for control of its relentless progression. The APBD patients, in this study, experienced stabilization of disease progression, and limited functional improvement in most patients with dietary triheptanoin. Due to a plateau in clinical improvement, the reduced plasma creatinine and methionine levels prompted evaluation of other plasma methylation intermediates in this complex integrated pathway system: decreased S-adenosylmethionine (SAM) (p<0.002), increased S-adenosylhomocysteine (p<0.001), elevated creatine (p=0.001) and increased free choline (p<0.001). Plasma levels of homocysteine and guanidinoacetate were normal. Impaired metabolism of choline and creatine may relate to the progressive dysmyelination and progressive muscle weakness associated with APBD. The partial deficiency of GBE appears to produce a secondary energy deficit possibly related to inadequate reserves of normal glycogen for efficient degradation to free glucose. Dysfunctional regulation of glycogen synthase (GS) may result in continued synthesis and deposition of polyglucosan bodies. This investigation has demonstrated, for the first time, arrest of clinical deterioration with limited functional recovery with triheptanoin diet therapy and the existence of significant derangement of methylation pathways that, when corrected, may lead to even greater therapeutic benefits.
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Affiliation(s)
- Charles R Roe
- Baylor Research Institute, Institute of Metabolic Disease, Baylor University Medical Center, Dallas, TX 75226, United States.
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Pekary AE, Stevens SA, Blood JD, Sattin A. Rapid modulation of TRH and TRH-like peptide release in rat brain, pancreas, and testis by a GSK-3beta inhibitor. Peptides 2010; 31:1083-93. [PMID: 20338209 DOI: 10.1016/j.peptides.2010.03.020] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/06/2010] [Accepted: 03/15/2010] [Indexed: 02/06/2023]
Abstract
Antidepressants have been shown to be neuroprotective and able to reverse damage to glia and neurons. Thyrotropin-releasing hormone (TRH) is an endogenous antidepressant-like neuropeptide that reduces the expression of glycogen synthase kinase-3beta (GSK-3beta), an enzyme that hyperphosphorylates tau and is implicated in bipolar disorder, diabetes and Alzheimer's disease. In order to understand the potential role of GSK-3beta in the modulation of depression by TRH and TRH-like peptides and the therapeutic potential of GSK-3beta inhibitors for neuropsychiatric and metabolic diseases, young adult male Sprague-Dawley (SD) rats were (a) injected ip with 1.8mg/kg of GSK-3beta inhibitor VIII (GSKI) and sacrificed 0, 2, 4, 6, and 8h later or (b) injected with 0, 0.018, 0.18 or 1.8mg/kg GSKI and bled 4h later. Levels of TRH and TRH-like peptides were measured in various brain regions involved in mood regulation, pancreas and reproductive tissues. Large, 3-15-fold, increases of TRH and TRH-like peptide levels in cerebellum, for example, as well as other brain regions were noted at 2 and 4h. In contrast, a nearly complete loss of TRH and TRH-like peptides from testis within 2h and pancreas by 4h following GSKI injection was observed. We have previously reported similar acute effects of corticosterone in brain and peripheral tissues. Incubation of a decapsulated rat testis with either GSKI or corticosterone accelerated release of TRH, and TRH-like peptides. Glucocorticoids, via inhibition of GSK3-beta activity, may thus be involved in the inhibition of TRH and TRH-like peptide release in brain, thereby contributing to the depressogenic effect of this class of steroids. Corticosterone-induced acceleration of release of these peptides from testis may contribute to the decline in reproductive function and redirection of energy needed during life-threatening emergencies. These contrasting effects of glucocorticoid on peptide release appear to be mediated by GSK-3beta.
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Affiliation(s)
- Albert Eugene Pekary
- Research Services, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, United States.
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Ciaraldi TP, Carter L, Mudaliar S, Henry RR. GSK-3beta and control of glucose metabolism and insulin action in human skeletal muscle. Mol Cell Endocrinol 2010; 315:153-8. [PMID: 19505532 PMCID: PMC2819161 DOI: 10.1016/j.mce.2009.05.020] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/14/2009] [Accepted: 05/27/2009] [Indexed: 12/27/2022]
Abstract
The involvement of the beta-isoform of glycogen synthase kinase (GSK-3) in glucose metabolism and insulin action was investigated in cultured human skeletal muscle cells. A 60% reduction in GSK-3beta protein expression was attained by treatment with siRNA; GSK-3alpha expression was unaltered. GSK-3beta knockdown did not influence total glycogen synthase (GS) activity, but increased the phosphorylation-dependent activity (fractional velocity-FV) in the basal state. Insulin responsiveness of GSFV was doubled by GSK-3beta knockdown (p<0.05). Basal rates of glucose uptake (GU) were not significantly influenced by GSK-3beta knockdown, while insulin stimulation of GU was increased. Improvements in insulin action on GS and GU did not involve changes in protein expression of either IRS-1 or Akt 1/2. Maximal insulin stimulation of phosphorylation of Akt was unaltered by GSK-3beta knockdown. Unlike GSK-3alpha, GSK-3beta directly regulates both GS activity in the absence of added insulin and through control of insulin action.
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Affiliation(s)
- T P Ciaraldi
- Veterans Affairs San Diego Healthcare System and Department of Medicine, University of California, 3350 La Jolla Village Drive, San Diego, CA 92161, USA
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Dynamic telomerase gene suppression via network effects of GSK3 inhibition. PLoS One 2009; 4:e6459. [PMID: 19649288 PMCID: PMC2714081 DOI: 10.1371/journal.pone.0006459] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2009] [Accepted: 06/30/2009] [Indexed: 01/15/2023] Open
Abstract
Background Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. Methodology/Principal Findings In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3′-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFκB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. Conclusions/Significance Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting.
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Tsai CC, Kai JI, Huang WC, Wang CY, Wang Y, Chen CL, Fang YT, Lin YS, Anderson R, Chen SH, Tsao CW, Lin CF. Glycogen Synthase Kinase-3β Facilitates IFN-γ-Induced STAT1 Activation by Regulating Src Homology-2 Domain-Containing Phosphatase 2. THE JOURNAL OF IMMUNOLOGY 2009; 183:856-64. [DOI: 10.4049/jimmunol.0804033] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
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Rayasam GV, Tulasi VK, Sodhi R, Davis JA, Ray A. Glycogen synthase kinase 3: more than a namesake. Br J Pharmacol 2009; 156:885-98. [PMID: 19366350 DOI: 10.1111/j.1476-5381.2008.00085.x] [Citation(s) in RCA: 376] [Impact Index Per Article: 23.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, tau protein and beta catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target.
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Affiliation(s)
- Geetha Vani Rayasam
- Department of Pharmacology, Research & Development (R&D III), Ranbaxy Research Labs, Gurgaon, Haryana, India.
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Xiong Q, Deng CY, Chai J, Jiang SW, Xiong YZ, Li FE, Zheng R. Knockdown of endogenous SKIP gene enhanced insulin-induced glycogen synthesis signaling in differentiating C2C12 myoblasts. BMB Rep 2009; 42:119-24. [DOI: 10.5483/bmbrep.2009.42.2.119] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
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Seo YH, Jung HJ, Shin HT, Kim YM, Yim H, Chung HY, Lim IK, Yoon G. Enhanced glycogenesis is involved in cellular senescence via GSK3/GS modulation. Aging Cell 2008; 7:894-907. [PMID: 18782348 DOI: 10.1111/j.1474-9726.2008.00436.x] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Glycogen biogenesis and its response to physiological stimuli have often been implicated in age-related diseases. However, their direct relationships to cell senescence and aging have not been clearly elucidated. Here, we report the central involvement of enhanced glycogenesis in cellular senescence. Glycogen accumulation, glycogen synthase (GS) activation, and glycogen synthase kinase 3 (GSK3) inactivation commonly occurred in diverse cellular senescence models, including the liver tissues of aging F344 rats. Subcytotoxic concentrations of GSK3 inhibitors (SB415286 and LiCl) were sufficient to induce cellular senescence with increased glycogenesis. Interestingly, the SB415286-induced glycogenesis was irreversible, as were increased levels of reactive oxygen species and gain of senescence phenotypes. Blocking GSK3 activity using siRNA or dominant negative mutant (GSK3beta-K85A) also effectively induced senescence phenotypes, and GS knock-down significantly attenuated the stress-induced senescence phenotypes. Taken together, these results clearly demonstrate that augmented glycogenesis is not only common, but is also directly linked to cellular senescence and aging, suggesting GSK3 and GS as novel modulators of senescence, and providing new insight into the metabolic backgrounds of aging and aging-related pathogenesis.
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Affiliation(s)
- Yong-Hak Seo
- Department of Molecular Science and Technology, The Graduate School, Ajou University, Suwon 443-721, Korea
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Kotliarova S, Pastorino S, Kovell LC, Kotliarov Y, Song H, Zhang W, Bailey R, Maric D, Zenklusen JC, Lee J, Fine HA. Glycogen synthase kinase-3 inhibition induces glioma cell death through c-MYC, nuclear factor-kappaB, and glucose regulation. Cancer Res 2008; 68:6643-51. [PMID: 18701488 PMCID: PMC2585745 DOI: 10.1158/0008-5472.can-08-0850] [Citation(s) in RCA: 223] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, is involved in diverse cellular processes ranging from nutrient and energy homeostasis to proliferation and apoptosis. Its role in glioblastoma multiforme has yet to be elucidated. We identified GSK3 as a regulator of glioblastoma multiforme cell survival using microarray analysis and small-molecule and genetic inhibitors of GSK3 activity. Various molecular and genetic approaches were then used to dissect out the molecular mechanisms responsible for GSK3 inhibition-induced cytotoxicity. We show that multiple small molecular inhibitors of GSK3 activity and genetic down-regulation of GSK3alpha/beta significantly inhibit glioma cell survival and clonogenicity. The potency of the cytotoxic effects is directly correlated with decreased enzyme activity-activating phosphorylation of GSK3alpha/beta Y276/Y216 and with increased enzyme activity inhibitory phosphorylation of GSK3alpha S21. Inhibition of GSK3 activity results in c-MYC activation, leading to the induction of Bax, Bim, DR4/DR5, and tumor necrosis factor-related apoptosis-inducing ligand expression and subsequent cytotoxicity. Additionally, down-regulation of GSK3 activity results in alteration of intracellular glucose metabolism resulting in dissociation of hexokinase II from the outer mitochondrial membrane with subsequent mitochondrial destabilization. Finally, inhibition of GSK3 activity causes a dramatic decrease in intracellular nuclear factor-kappaB activity. Inhibition of GSK3 activity results in c-MYC-dependent glioma cell death through multiple mechanisms, all of which converge on the apoptotic pathways. GSK3 may therefore be an important therapeutic target for gliomas. Future studies will further define the optimal combinations of GSK3 inhibitors and cytotoxic agents for use in gliomas and other cancers.
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Affiliation(s)
- Svetlana Kotliarova
- Neuro-Oncology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA
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Wang ZQ, Ribnicky D, Zhang XH, Raskin I, Yu Y, Cefalu WT. Bioactives of Artemisia dracunculus L enhance cellular insulin signaling in primary human skeletal muscle culture. Metabolism 2008; 57:S58-64. [PMID: 18555856 PMCID: PMC2981033 DOI: 10.1016/j.metabol.2008.04.003] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
An alcoholic extract of Artemisia dracunculus L (PMI 5011) has been shown to decrease glucose and improve insulin levels in animal models, suggesting an ability to enhance insulin sensitivity. We sought to assess the cellular mechanism by which this botanical affects carbohydrate metabolism in primary human skeletal muscle culture. We measured basal and insulin-stimulated glucose uptake, glycogen accumulation, phosphoinositide 3 (PI-3) kinase activity, and Akt phosphorylation in primary skeletal muscle culture from subjects with type 2 diabetes mellitus incubated with or without various concentrations of PMI 5011. We also analyzed the abundance of insulin receptor signaling proteins, for example, IRS-1, IRS-2, and PI-3 kinase. Glucose uptake was significantly increased in the presence of increasing concentrations of PMI 5011. In addition, glycogen accumulation, observed to be decreased with increasing free fatty acid levels, was partially restored with PMI 5011. PMI 5011 treatment did not appear to significantly affect protein abundance for IRS-1, IRS-2, PI-3 kinase, Akt, insulin receptor, or Glut-4. However, PMI 5011 significantly decreased levels of a specific protein tyrosine phosphatase, that is, PTP1B. Time course studies confirmed that protein abundance of PTP1B decreases in the presence of PMI 5011. The cellular mechanism of action to explain the effects by which an alcoholic extract of A dracunculus L improves carbohydrate metabolism on a clinical level may be secondary to enhancing insulin receptor signaling and modulating levels of a specific protein tyrosine phosphatase, that is, PTP1B.
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Affiliation(s)
- Zhong Q. Wang
- Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA 70808, USA
| | - David Ribnicky
- Biotech Center, Rutgers University, New Brunswick, NJ 08901, USA
| | - Xian H. Zhang
- Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA 70808, USA
| | - Ilya Raskin
- Biotech Center, Rutgers University, New Brunswick, NJ 08901, USA
| | - Yongmei Yu
- Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA 70808, USA
| | - William T. Cefalu
- Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA 70808, USA
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Underwood KR, Tong J, Zhu MJ, Shen QW, Means WJ, Ford SP, Paisley SI, Hess BW, Du M. Relationship between kinase phosphorylation, muscle fiber typing, and glycogen accumulation in longissimus muscle of beef cattle with high and low intramuscular fat. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2007; 55:9698-9703. [PMID: 17935292 DOI: 10.1021/jf071573z] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/25/2023]
Abstract
The objective of this study was to examine the association of adenosine monophosphate (AMP)-activated protein kinase (AMPK) with glycogen content in bovine muscle and their links with intramuscular fat (IMF) and muscle fiber type composition. Five steers with high intramuscular fat (High IMF, IMF content is 5.71 +/- 0.36%) and five steers with low intramuscular fat (Low IMF, IMF content is 2.09 +/- 0.19%) in the longissimus thoracis muscle (LM) were selected for immunoblotting, glycogen, and myofiber type composition analyses. The glycogen content was higher in Low IMF muscle than in High IMF muscle (1.07 +/- 0.07 versus 0.85 +/- 0.08 g/100 g muscle, P < 0.05). Phosphorylation of the AMPK alpha subunit at Thr 172, which is correlated with its activity, was lower (P < 0.05) in High IMF compared to Low IMF. In agreement with the lower AMPK phosphorylation in High IMF muscle, the phosphorylation of acetyl-CoA carboxylase (ACC) was also lower (P < 0.05) in High IMF muscle than in Low IMF muscle. Glycogen synthase kinase 3 (GSK3) down-regulates glycogen synthesis through phosphorylation of glycogen synthase. The phosphorylation of GSK3 in High IMF was lower (P < 0.05) than that in Low IMF, which should down-regulate glycogen synthase activity and reduce the glycogen content in High IMF beef. Type IIB myosin isoform was absent in beef muscle. No noticeable difference in myosin isoform composition was observed between Low and High IMF muscle. In summary, High IMF cattle had lower LM glycogen levels than low IMF cattle, and AMPK activity was less in High IMF than in Low IMF cattle. The difference in glycogen content between Low and High IMF muscle was not correlated with muscle fiber composition. This data shows that LM lipid and glycogen metabolisms are affected by AMPK activity. Thus, AMPK may be a molecular target to alter IMF and glycogen levels in beef muscle.
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Affiliation(s)
- Keith R Underwood
- Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071, USA
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MacAulay K, Doble BW, Patel S, Hansotia T, Sinclair EM, Drucker DJ, Nagy A, Woodgett JR. Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism. Cell Metab 2007; 6:329-37. [PMID: 17908561 DOI: 10.1016/j.cmet.2007.08.013] [Citation(s) in RCA: 240] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/12/2007] [Revised: 07/14/2007] [Accepted: 08/24/2007] [Indexed: 01/21/2023]
Abstract
Glycogen synthase kinase 3 comprises two isoforms (GSK-3alpha and GSK-3beta) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3alpha. Unlike GSK-3beta mutants, which die before birth, GSK-3alpha knockout (GSK-3alpha KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3alpha KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3beta phosphorylation was higher in GSK-3alpha KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3alpha KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes.
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Affiliation(s)
- Katrina MacAulay
- Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada
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Cleasby ME, Reinten TA, Cooney GJ, James DE, Kraegen EW. Functional Studies of Akt Isoform Specificity in Skeletal Muscle in Vivo; Maintained Insulin Sensitivity Despite Reduced Insulin Receptor Substrate-1 Expression. Mol Endocrinol 2007; 21:215-28. [PMID: 17021050 DOI: 10.1210/me.2006-0154] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AbstractThe phosphoinositide 3-kinase/Akt pathway is thought to be essential for normal insulin action and glucose metabolism in skeletal muscle and has been shown to be dysregulated in insulin resistance. However, the specific roles of and signaling pathways triggered by Akt isoforms have not been fully assessed in muscle in vivo. We overexpressed constitutively active (ca-) Akt-1 or Akt-2 constructs in muscle using in vivo electrotransfer and, after 1 wk, assessed the roles of each isoform on glucose metabolism and fiber growth. We achieved greater than 2.5-fold increases in total Ser473 phosphorylation in muscles expressing ca-Akt-1 and ca-Akt-2, respectively. Both isoforms caused hypertrophy of muscle fibers, consistent with increases in p70S6kinase phosphorylation, and a 60% increase in glycogen accumulation, although only Akt-1 increased glycogen synthase kinase-3β phosphorylation. Akt-2, but not Akt-1, increased basal glucose uptake (by 33%, P = 0.004) and incorporation into glycogen and lipids, suggesting a specific effect on glucose transport. Consistent with this, short hairpin RNA-mediated silencing of Akt-2 caused reductions in glycogen storage and glucose uptake. Consistent with Akt-mediated insulin receptor substrate 1 (IRS-1) degradation, we observed approximately 30% reductions in IRS-1 protein in muscle overexpressing ca-Akt-1 or ca-Akt-2. Despite this, we observed no decrease in insulin-stimulated glucose uptake. Furthermore, a 68% reduction in IRS-1 levels induced using short hairpin RNAs targeting IRS-1 also did not affect glucose disposal after a glucose load. These data indicate distinct roles for Akt-1 and Akt-2 in muscle glucose metabolism and that moderate reductions in IRS-1 expression do not result in the development of insulin resistance in skeletal muscle in vivo.
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Affiliation(s)
- Mark E Cleasby
- Diabetes and Obesity Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia.
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Fernández-Novell JM, Rodríguez-Gil JE, Barberà A, Guinovart JJ. Lithium ions increase hepatic glycogen synthase stability through a proteasome-related mechanism. Arch Biochem Biophys 2007; 457:29-34. [PMID: 17125726 DOI: 10.1016/j.abb.2006.10.009] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2006] [Revised: 10/06/2006] [Accepted: 10/12/2006] [Indexed: 11/18/2022]
Abstract
Incubation of rat hepatocytes with LiCl resulted in an overall increase in the activity ratio of glycogen synthase (GS), concomitantly with a decrease in active GS kinase-3 levels. GS total activity was also increased in a dose- and time-dependent manner. This latter effect correlated with the amount of immunoreactive enzyme determined by immunoblotting. Cycloheximide and actinomycin-D did not modify LiCl action on GS activity. Lithium ions did not induce any changes in GS mRNA levels. Furthermore, the increase in the total amount of GS induced by LiCl was further augmented after addition of a specific, calpain and proteasome inhibitor. Our results indicate that LiCl increases hepatocyte GS activity through increasing both the activation state of the enzyme and its cellular content. This latter increase is mediated through a modification of the proteasome-regulated proteolytic pathway of the enzyme.
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Affiliation(s)
- Josep M Fernández-Novell
- Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Avgda. Diagonal 645, Edifici nou planta-1, E-08028 Barcelona, Spain.
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Yamamoto DL, Hutchinson DS, Bengtsson T. Beta(2)-Adrenergic activation increases glycogen synthesis in L6 skeletal muscle cells through a signalling pathway independent of cyclic AMP. Diabetologia 2007; 50:158-67. [PMID: 17119919 DOI: 10.1007/s00125-006-0484-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/27/2006] [Accepted: 09/01/2006] [Indexed: 01/05/2023]
Abstract
AIMS/HYPOTHESIS In skeletal muscle, the storage of glycogen by insulin is regulated by glycogen synthase, which is regulated by glycogen synthase kinase 3 (GSK3). Here we examined whether adrenergic receptor activation, which can increase glucose uptake, regulates glycogen synthesis in L6 skeletal muscle cells. METHODS We used L6 cells and measured glycogen synthesis (as incorporation of D: -[U-(14)C]glucose into glycogen) and GSK3 phosphorylation following adrenergic activation. RESULTS Insulin (negative logarithm of median effective concentration [pEC(50)] 8.2 +/- 0.3) and the beta-adrenergic agonist isoprenaline (pEC(50) 7.5 +/- 0.3) induced a twofold increase in glycogen synthesis in a concentration-dependent manner. The alpha(1)-adrenergic agonist cirazoline and alpha(2)-adrenergic agonist clonidine had no effect. Both insulin and isoprenaline phosphorylated GSK3. The beta-adrenergic effect on glycogen synthesis is mediated by beta(2)-adrenoceptors and not beta(1)-/beta(3)-adrenoceptors, and was not mimicked by 8-bromo-cyclic AMP or cholera toxin, and also was insensitive to pertussis toxin, indicating no involvement of cyclic AMP or inhibitory G-protein (G(i)) signalling in the beta(2)-adrenergic effect on glycogen synthesis. 12-O-tetra-decanoylphorbol-13-acetate (TPA) increased glycogen synthesis 2.5-fold and phosphorylated GSK3 fourfold. Inhibition of protein kinase C (PKC) isoforms with 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrollo(3,4-c)-carbazole (Gö6976; inhibits conventional and novel PKCs) or 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983; inhibits conventional, novel and atypical PKCs) inhibited the stimulatory TPA effect, but did not significantly inhibit glycogen synthesis mediated by insulin or isoprenaline. Inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin inhibited the effects of insulin and isoprenaline on glycogen synthesis. CONCLUSIONS/INTERPRETATION These results demonstrate that in L6 skeletal muscle cells adrenergic stimulation through beta(2)-adrenoceptors, but not involving cyclic AMP or G(i), activates a PI3K pathway that stimulates glycogen synthesis through GSK3.
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Affiliation(s)
- D L Yamamoto
- Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, SE 10691, Stockholm, Sweden
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