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Domma AJ, Henderson LA, Goodrum FD, Moorman NJ, Kamil JP. Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins. J Virol 2023; 97:e0056323. [PMID: 37754763 PMCID: PMC10617551 DOI: 10.1128/jvi.00563-23] [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: 04/15/2023] [Accepted: 08/09/2023] [Indexed: 09/28/2023] Open
Abstract
IMPORTANCE Human cytomegalovirus (HCMV) requires inactivation of AKT to efficiently replicate, yet how AKT is shut off during HCMV infection has remained unclear. We show that UL38, an HCMV protein that activates mTORC1, is necessary and sufficient to destabilize insulin receptor substrate 1 (IRS1), a model insulin receptor substrate (IRS) protein. Degradation of IRS proteins in settings of excessive mTORC1 activity is an important mechanism for insulin resistance. When IRS proteins are destabilized, PI3K cannot be recruited to growth factor receptor complexes, and hence, AKT membrane recruitment, a rate limiting step in its activation, fails to occur. Despite its penchant for remodeling host cell signaling pathways, our results reveal that HCMV relies upon a cell-intrinsic negative regulatory feedback loop to inactivate AKT. Given that pharmacological inhibition of PI3K/AKT potently induces HCMV reactivation from latency, our findings also imply that the expression of UL38 activity must be tightly regulated within latently infected cells to avoid spontaneous reactivation.
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Affiliation(s)
- Anthony J. Domma
- Department of Microbiology and Immunology, Louisiana State University Health Sciences Center Shreveport, Shreveport, Louisiana, USA
| | - Lauren A. Henderson
- Department of Microbiology and Immunology, Louisiana State University Health Sciences Center Shreveport, Shreveport, Louisiana, USA
| | - Felicia D. Goodrum
- Department of Immunobiology, University of Arizona, Tucson, Arizona, USA
- Bio5 Institute, University of Arizona, Tucson, Arizona, USA
| | - Nathaniel J. Moorman
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Jeremy P. Kamil
- Department of Microbiology and Immunology, Louisiana State University Health Sciences Center Shreveport, Shreveport, Louisiana, USA
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Domma AJ, Goodrum FD, Moorman NJ, Kamil JP. Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.17.537203. [PMID: 37131605 PMCID: PMC10153195 DOI: 10.1101/2023.04.17.537203] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
The phosphoinositide 3-kinase (PI3K)/AKT pathway plays crucial roles in cell viability and protein synthesis and is frequently co-opted by viruses to support their replication. Although many viruses maintain high levels of AKT activity during infection, other viruses, such as vesicular stomatitis virus and human cytomegalovirus (HCMV), cause AKT to accumulate in an inactive state. To efficiently replicate, HCMV requires FoxO transcription factors to localize to the infected cell nucleus (Zhang et. al. mBio 2022), a process directly antagonized by AKT. Therefore, we sought to investigate how HCMV inactivates AKT to achieve this. Subcellular fractionation and live cell imaging studies indicated that AKT failed to recruit to membranes upon serum-stimulation of infected cells. However, UV-inactivated virions were unable to render AKT non-responsive to serum, indicating a requirement for de novo viral gene expression. Interestingly, we were able to identify that UL38 (pUL38), a viral activator of mTORC1, is required to diminish AKT responsiveness to serum. mTORC1 contributes to insulin resistance by causing proteasomal degradation of insulin receptor substrate (IRS) proteins, such as IRS1, which are necessary for the recruitment of PI3K to growth factor receptors. In cells infected with a recombinant HCMV disrupted for UL38 , AKT responsiveness to serum is retained and IRS1 is not degraded. Furthermore, ectopic expression of UL38 in uninfected cells induces IRS1 degradation, inactivating AKT. These effects of UL38 were reversed by the mTORC1 inhibitor, rapamycin. Collectively, our results demonstrate that HCMV relies upon a cell-intrinsic negative feedback loop to render AKT inactive during productive infection.
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Affiliation(s)
- Anthony J. Domma
- Department of Microbiology and Immunology, LSU Health Sciences Center Shreveport, Shreveport Louisiana, USA
| | - Felicia D. Goodrum
- Department of Immunobiology, University of Arizona, Tucson, AZ, USA
- Bio5 Institute, University of Arizona, Tucson, AZ, USA
| | - Nathaniel J. Moorman
- Department of Microbiology and Immunology, UNC Chapel Hill, Chapel Hill, NC, USA
| | - Jeremy P. Kamil
- Department of Microbiology and Immunology, LSU Health Sciences Center Shreveport, Shreveport Louisiana, USA
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3
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O'Reilly CL, Uranga S, Fluckey JD. Culprits or consequences: Understanding the metabolic dysregulation of muscle in diabetes. World J Biol Chem 2021; 12:70-86. [PMID: 34630911 PMCID: PMC8473417 DOI: 10.4331/wjbc.v12.i5.70] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Revised: 06/21/2021] [Accepted: 08/03/2021] [Indexed: 02/06/2023] Open
Abstract
The prevalence of type 2 diabetes (T2D) continues to rise despite the amount of research dedicated to finding the culprits of this debilitating disease. Skeletal muscle is arguably the most important contributor to glucose disposal making it a clear target in insulin resistance and T2D research. Within skeletal muscle there is a clear link to metabolic dysregulation during the progression of T2D but the determination of culprits vs consequences of the disease has been elusive. Emerging evidence in skeletal muscle implicates influential cross talk between a key anabolic regulatory protein, the mammalian target of rapamycin (mTOR) and its associated complexes (mTORC1 and mTORC2), and the well-described canonical signaling for insulin-stimulated glucose uptake. This new understanding of cellular signaling crosstalk has blurred the lines of what is a culprit and what is a consequence with regard to insulin resistance. Here, we briefly review the most recent understanding of insulin signaling in skeletal muscle, and how anabolic responses favoring anabolism directly impact cellular glucose disposal. This review highlights key cross-over interactions between protein and glucose regulatory pathways and the implications this may have for the design of new therapeutic targets for the control of glucoregulatory function in skeletal muscle.
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Affiliation(s)
| | - Selina Uranga
- Health and Kinesiology, Texas A&M University, TX 77843, United States
| | - James D Fluckey
- Health and Kinesiology, Texas A&M University, TX 77843, United States
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Disruption of the Phosphate Transporter Pit1 in Hepatocytes Improves Glucose Metabolism and Insulin Signaling by Modulating the USP7/IRS1 Interaction. Cell Rep 2016; 16:2736-2748. [PMID: 27568561 DOI: 10.1016/j.celrep.2016.08.012] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2016] [Revised: 06/02/2016] [Accepted: 08/03/2016] [Indexed: 01/07/2023] Open
Abstract
The liver plays a central role in whole-body lipid and glucose homeostasis. Increasing dietary fat intake results in increased hepatic fat deposition, which is associated with a risk for development of insulin resistance and type 2 diabetes. In this study, we demonstrate a role for the phosphate inorganic transporter 1 (PiT1/SLC20A1) in regulating metabolism. Specific knockout of Pit1 in hepatocytes significantly improved glucose tolerance and insulin sensitivity, enhanced insulin signaling, and decreased hepatic lipogenesis. We identified USP7 as a PiT1 binding partner and demonstrated that Pit1 deletion inhibited USP7/IRS1 dissociation upon insulin stimulation. This prevented IRS1 ubiquitination and its subsequent proteasomal degradation. As a consequence, delayed insulin negative feedback loop and sustained insulin signaling were observed. Moreover, PiT1-deficient mice were protected against high-fat-diet-induced obesity and diabetes. Our findings indicate that PiT1 has potential as a therapeutic target in the context of metabolic syndrome, obesity, and diabetes.
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Martin-Gronert MS, Fernandez-Twinn DS, Bushell M, Siddle K, Ozanne SE. Cell-autonomous programming of rat adipose tissue insulin signalling proteins by maternal nutrition. Diabetologia 2016; 59:1266-75. [PMID: 26965244 PMCID: PMC4861755 DOI: 10.1007/s00125-016-3905-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/14/2015] [Accepted: 02/03/2016] [Indexed: 01/10/2023]
Abstract
AIMS/HYPOTHESIS Individuals with a low birthweight have an increased risk of developing type 2 diabetes mellitus in adulthood. This is associated with peripheral insulin resistance. Here, we aimed to determine whether changes in insulin signalling proteins in white adipose tissue (WAT) can be detected prior to the onset of impaired glucose tolerance, determine whether these changes are cell-autonomous and identify the underlying mechanisms involved. METHODS Fourteen-month-old male rat offspring born to dams fed a standard protein (20%) diet or a low (8%) protein diet throughout gestation and lactation were studied. Fat distribution and adipocyte size were determined. Protein content and mRNA expression of key insulin signalling molecules were analysed in epididymal WAT and in pre-adipocytes that had undergone in vitro differentiation. RESULTS The offspring of low protein fed dams (LP offspring) had reduced visceral WAT mass, altered fat distribution and a higher percentage of small adipocytes in epididymal WAT. This was associated with reduced levels of IRS1, PI3K p110β, Akt1 and PKCζ proteins and of phospho-Akt Ser473. Corresponding mRNA transcript levels were unchanged. Similarly, in vitro differentiated adipocytes from LP offspring showed reduced protein levels of IRβ, IRS1, PI3K p85α and p110β subunits, and Akt1. Levels of Akt Ser473 and IRS1 Tyr612 phosphorylation were reduced, while IRS1 Ser307 phosphorylation was increased. CONCLUSIONS/INTERPRETATION Maternal protein restriction during gestation and lactation changes the distribution and morphology of WAT and reduces the levels of key insulin signalling proteins in the male offspring. This phenotype is retained in in vitro differentiated adipocytes, suggesting that programming occurs via cell-autonomous mechanism(s).
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Affiliation(s)
- Malgorzata S Martin-Gronert
- University of Cambridge Metabolic Research Laboratories and MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Box 289, Cambridge, CB2 OQQ, UK.
| | - Denise S Fernandez-Twinn
- University of Cambridge Metabolic Research Laboratories and MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Box 289, Cambridge, CB2 OQQ, UK
| | - Martin Bushell
- MRC Toxicology Unit, University of Leicester, Hodgkin Building, Leicester, UK
| | - Kenneth Siddle
- University of Cambridge Metabolic Research Laboratories and MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Box 289, Cambridge, CB2 OQQ, UK
| | - Susan E Ozanne
- University of Cambridge Metabolic Research Laboratories and MRC Metabolic Diseases Unit, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Box 289, Cambridge, CB2 OQQ, UK
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6
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Kusminski CM, Gallardo-Montejano VI, Wang ZV, Hegde V, Bickel PE, Dhurandhar NV, Scherer PE. E4orf1 induction in adipose tissue promotes insulin-independent signaling in the adipocyte. Mol Metab 2015; 4:653-64. [PMID: 26500839 PMCID: PMC4588421 DOI: 10.1016/j.molmet.2015.07.004] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2015] [Revised: 07/08/2015] [Accepted: 07/13/2015] [Indexed: 12/21/2022] Open
Abstract
Background/Purpose Type 2 diabetes remains a worldwide epidemic with major pathophysiological changes as a result of chronic insulin resistance. Insulin regulates numerous biochemical pathways related to carbohydrate and lipid metabolism. Methods We have generated a novel mouse model that allows us to constitutively activate, in an inducible fashion, the distal branch of the insulin signaling transduction pathway specifically in adipocytes. Results Using the adenoviral 36 E4orf1 protein, we chronically stimulate locally the Ras-ERK-MAPK signaling pathway. At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge. Despite overlapping glucose tolerance curves, there is a reduced requirement for insulin action under these conditions. The mice further exhibit reduced circulating adiponectin levels that ultimately lead to impaired lipid clearance, and inflamed and fibrotic white adipose tissues. Nevertheless, they are protected from diet-induced hepatic steatosis. As we observe constitutively elevated p-Akt levels in the adipocytes, even under conditions of low insulin levels, this pinpoints enhanced Ras-ERK-MAPK signaling in transgenic adipocytes as a potential alternative route to bypass proximal insulin signaling events. Conclusion We conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.
Inducible activation of the distal branch of the insulin pathway in adipocytes. Insulin-sparing characteristics during glucose tolerance testing. Chronic activation of the distal Ras-ERK-MAPK signaling pathway. Reduced body-weight during metabolic challenge. Preserved carbohydrate metabolism at the expense of lipid metabolism.
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Affiliation(s)
- Christine M Kusminski
- Touchstone Diabetes Center, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Violeta I Gallardo-Montejano
- Division of Endocrinology, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Zhao V Wang
- Touchstone Diabetes Center, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Vijay Hegde
- Department of Infection and Obesity Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA, USA
| | - Perry E Bickel
- Division of Endocrinology, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Nikhil V Dhurandhar
- Department of Infection and Obesity Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA, USA
| | - Philipp E Scherer
- Touchstone Diabetes Center, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, USA ; Department of Cell Biology, The University of Texas Southwestern Medical Center, Dallas, TX, USA
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7
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Chai SP, Fong JC. Synergistic induction of insulin resistance by endothelin-1 and cAMP in 3T3-L1 adipocytes. Biochim Biophys Acta Mol Basis Dis 2015; 1852:2048-55. [PMID: 26143144 DOI: 10.1016/j.bbadis.2015.06.026] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2015] [Revised: 05/30/2015] [Accepted: 06/25/2015] [Indexed: 12/27/2022]
Abstract
Both endothelin-1 (ET-1) and cAMP are implicated for inducing insulin resistance. Since we have shown previously that there is a crosstalk between ET-1 and cAMP signaling pathways in regulating glucose uptake in 3T3-L1 adipocytes, we extended our investigation in this study on whether they may have a synergistic effect on inducing insulin resistance. Our results showed that it was indeed the case. Insulin-stimulated glucose uptake, phosphorylation of PKB, IRS-1-associated PI3K, and IRS-1 tyrosine phosphorylation were all inhibited by ET-1 and 8-bromo cAMP in a synergistic manner. IRS-1 protein levels were similarly decreased by ET-1 and 8-bromo cAMP, attributable to suppressed mRNA expression. In addition, after correction for the loss in IRS-1 protein, the inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation or IRS-1-associated PI3K was mainly caused by cAMP. Moreover, whereas IRS-2 protein levels were increased by cAMP or ET-1 plus cAMP, insulin-stimulated IRS-2-associated PI3K activities were abolished by both treatments. Furthermore, ET-1 and β-adrenergic agonists had similar synergistic inhibition on insulin-stimulated glucose uptake. In conclusion, we have shown that ET-1 and cAMP may synergistically induce insulin resistance in adipocytes via inhibiting IRS-1 expression as well as insulin-stimulated IRS-1/IRS-2 activities.
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Affiliation(s)
- Shin-Pei Chai
- Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan, ROC
| | - Jim C Fong
- Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan, ROC.
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Tan SX, Fisher-Wellman KH, Fazakerley DJ, Ng Y, Pant H, Li J, Meoli CC, Coster ACF, Stöckli J, James DE. Selective insulin resistance in adipocytes. J Biol Chem 2015; 290:11337-48. [PMID: 25720492 DOI: 10.1074/jbc.m114.623686] [Citation(s) in RCA: 81] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2014] [Indexed: 12/14/2022] Open
Abstract
Aside from glucose metabolism, insulin regulates a variety of pathways in peripheral tissues. Under insulin-resistant conditions, it is well known that insulin-stimulated glucose uptake is impaired, and many studies attribute this to a defect in Akt signaling. Here we make use of several insulin resistance models, including insulin-resistant 3T3-L1 adipocytes and fat explants prepared from high fat-fed C57BL/6J and ob/ob mice, to comprehensively distinguish defective from unaffected aspects of insulin signaling and its downstream consequences in adipocytes. Defective regulation of glucose uptake was observed in all models of insulin resistance, whereas other major actions of insulin such as protein synthesis and anti-lipolysis were normal. This defect corresponded to a reduction in the maximum response to insulin. The pattern of change observed for phosphorylation in the Akt pathway was inconsistent with a simple defect at the level of Akt. The only Akt substrate that showed consistently reduced phosphorylation was the RabGAP AS160 that regulates GLUT4 translocation. We conclude that insulin resistance in adipose tissue is highly selective for glucose metabolism and likely involves a defect in one of the components regulating GLUT4 translocation to the cell surface in response to insulin.
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Affiliation(s)
- Shi-Xiong Tan
- From the Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales 2010, Australia
| | - Kelsey H Fisher-Wellman
- From the Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales 2010, Australia
| | | | - Yvonne Ng
- From the Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales 2010, Australia
| | - Himani Pant
- From the Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales 2010, Australia
| | - Jia Li
- From the Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales 2010, Australia
| | - Christopher C Meoli
- From the Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales 2010, Australia, the Charles Perkins Centre, School of Molecular Biosciences and
| | - Adelle C F Coster
- the School of Mathematics and Statistics, University of New South Wales, Sydney, New South Wales 2052, Australia
| | | | - David E James
- the Charles Perkins Centre, School of Molecular Biosciences and the School of Medicine, University of Sydney, New South Wales 2006, Australia, and
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Wiza C, Herzfeld de Wiza D, Nascimento EBM, Lehr S, Al-Hasani H, Ouwens DM. Knockdown of PRAS40 inhibits insulin action via proteasome-mediated degradation of IRS1 in primary human skeletal muscle cells. Diabetologia 2013; 56:1118-28. [PMID: 23460019 DOI: 10.1007/s00125-013-2861-9] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/26/2012] [Accepted: 01/23/2013] [Indexed: 10/27/2022]
Abstract
AIMS/HYPOTHESIS The proline-rich Akt substrate of 40 kDa (PRAS40) is a component of the mammalian target of rapamycin complex 1 (mTORC1) and among the most prominent Akt substrates in skeletal muscle. Yet the cellular functions of PRAS40 are incompletely defined. This study assessed the function of PRAS40 in insulin action in primary human skeletal muscle cells (hSkMC). METHODS Insulin action was examined in hSkMC following small interfering RNA-mediated silencing of PRAS40 (also known as AKT1S1) under normal conditions and following chemokine-induced insulin resistance. RESULTS PRAS40 knockdown (PRAS40-KD) in hSkMC decreased insulin-mediated phosphorylation of Akt by 50% (p < 0.05) as well as of the Akt substrates glycogen synthase kinase 3 (40%) and tuberous sclerosis complex 2 (32%) (both p < 0.05). Furthermore, insulin-stimulated glucose uptake was reduced by 20% in PRAS40-KD myotubes (p < 0.05). Exposing PRAS40-KD myotubes to chemokines caused no additional deterioration of insulin action. PRAS40-KD further reduced insulin-mediated phosphorylation of the mTORC1-regulated proteins p70S6 kinase (p70S6K) (47%), S6 (43%), and eukaryotic elongation 4E-binding protein 1 (100%), as well as protein levels of growth factor receptor bound protein 10 (35%) (all p < 0.05). The inhibition of insulin action in PRAS40-KD myotubes was associated with a reduction in IRS1 protein levels (60%) (p < 0.05), and was reversed by pharmacological proteasome inhibition. Accordingly, expression of the genes encoding E3-ligases F-box protein 32 (also known as atrogin-1) and muscle RING-finger protein-1 and activity of the proteasome was elevated in PRAS40-KD myotubes. CONCLUSIONS/INTERPRETATION Inhibition of insulin action in PRAS40-KD myotubes was found to associate with IRS1 degradation promoted by increased proteasome activity rather than hyperactivation of the p70S6K-negative-feedback loop. These findings identify PRAS40 as a modulator of insulin action.
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MESH Headings
- Adaptor Proteins, Signal Transducing/antagonists & inhibitors
- Adaptor Proteins, Signal Transducing/genetics
- Adaptor Proteins, Signal Transducing/metabolism
- Cells, Cultured
- Chemokine CCL2/metabolism
- Chemokines/metabolism
- Down-Regulation/drug effects
- Female
- Gene Silencing
- Humans
- Hypoglycemic Agents/pharmacology
- Insulin Receptor Substrate Proteins/metabolism
- Insulin Resistance
- Insulin, Regular, Pork/pharmacology
- Intercellular Signaling Peptides and Proteins
- Male
- Muscle, Skeletal/cytology
- Muscle, Skeletal/drug effects
- Muscle, Skeletal/immunology
- Muscle, Skeletal/metabolism
- Phosphorylation/drug effects
- Proteasome Endopeptidase Complex/drug effects
- Proteasome Endopeptidase Complex/metabolism
- Protein Processing, Post-Translational/drug effects
- Proteolysis/drug effects
- Proto-Oncogene Proteins c-akt/metabolism
- RNA, Small Interfering
- Recombinant Proteins/metabolism
- Up-Regulation/drug effects
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Affiliation(s)
- C Wiza
- Institute for Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Auf´m Hennekamp 65, 40225 Düsseldorf, Germany
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Tan SX, Ng Y, Meoli CC, Kumar A, Khoo PS, Fazakerley DJ, Junutula JR, Vali S, James DE, Stöckli J. Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes. J Biol Chem 2011; 287:6128-38. [PMID: 22207758 PMCID: PMC3307283 DOI: 10.1074/jbc.m111.318238] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5–22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease.
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Affiliation(s)
- Shi-Xiong Tan
- Diabetes and Obesity Research Program, The Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, New South Wales 2010, Australia
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Octreotide-treated diabetes accompanied by endogenous hyperinsulinemic hypoglycemia and protein-losing gastroenteropathy. Case Rep Med 2011; 2011:381203. [PMID: 21826148 PMCID: PMC3150201 DOI: 10.1155/2011/381203] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2011] [Revised: 05/11/2011] [Accepted: 06/14/2011] [Indexed: 01/24/2023] Open
Abstract
Occurrence of hypoglycemia in diabetes patients is very rare. We report here a case of frequent hypoglycemic attacks caused by inappropriate endogenous hyperinsulinemia in a female patient with poorly controlled diabetes and protein-losing gastroenteropathy. The blood glucose profiles of the patient were unstable. Results of the fasting test performed to investigate the cause of hypoglycemia suggested endogenous hyperinsulinism. Repeated selective arterial calcium injection tests suggested that hyperinsulinemia might be extrapancreatic in origin. However, efforts to detect a responsible lesion such as insulinoma were unsuccessful. Octreotide was used for the treatment of hypoglycemia and protein-losing gastroenteropathy. After treatment, although her leg edema caused by hypoalbuminemia persisted, hypoglycemia almost disappeared.
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12
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Randriamboavonjy V, Fleming I. The Role of Calpain in Diabetes-Associated Platelet Hyperactivation. CARDIOVASCULAR PHARMACOLOGY - HEART AND CIRCULATION 2010; 59:235-57. [DOI: 10.1016/s1054-3589(10)59008-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
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13
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Wang Y, Nishina PM, Naggert JK. Degradation of IRS1 leads to impaired glucose uptake in adipose tissue of the type 2 diabetes mouse model TALLYHO/Jng. J Endocrinol 2009; 203:65-74. [PMID: 19587264 PMCID: PMC2853731 DOI: 10.1677/joe-09-0026] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
The TALLYHO/Jng (TH) mouse strain is a polygenic model for type 2 diabetes (T2D) characterized by moderate obesity, impaired glucose tolerance and uptake, insulin resistance, and hyperinsulinemia. The goal of this study was to elucidate the molecular mechanisms responsible for the reduced glucose uptake and insulin resistance in the adipose tissue of this model. The translocation and localization of glucose transporter 4 (GLUT4) to the adipocyte plasma membrane were impaired in TH mice compared to control C57BL6/J (B6) mice. These defects were associated with decreased GLUT4 protein, reduced phosphatidylinositol 3-kinase activity, and alterations in the phosphorylation status of insulin receptor substrate 1 (IRS1). Activation of c-Jun N-terminal kinase 1/2, which can phosphorylate IRS1 on Ser307, was significantly higher in TH mice compared with B6 controls. IRS1 protein but not mRNA levels was found to be lower in TH mice than controls. Immunoprecipitation with anti-ubiquitin and western blot analysis of IRS1 protein revealed increased total IRS1 ubiquitination in adipose tissue of TH mice. Suppressor of cytokine signaling 1, known to promote IRS1 ubiquitination and subsequent degradation, was found at significantly higher levels in TH mice compared with B6. Immunohistochemistry showed that IRS1 colocalized with the 20S proteasome in proteasomal structures in TH adipocytes, supporting the notion that IRS1 is actively degraded. Our findings suggest that increased IRS1 degradation and subsequent impaired GLUT4 mobilization play a role in the reduced glucose uptake in insulin resistant TH mice. Since low-IRS1 levels are often observed in human T2D, the TH mouse is an attractive model to investigate mechanisms of insulin resistance and explore new treatments.
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Affiliation(s)
- Yun Wang
- The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA
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14
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Wang ZQ, Floyd ZE, Qin J, Liu X, Yu Y, Zhang XH, Wagner JD, Cefalu WT. Modulation of skeletal muscle insulin signaling with chronic caloric restriction in cynomolgus monkeys. Diabetes 2009; 58:1488-98. [PMID: 19336678 PMCID: PMC2699875 DOI: 10.2337/db08-0977] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
OBJECTIVE Caloric restriction (CR) has been shown to retard aging processes, extend maximal life span, and consistently increase insulin action in experimental animals. The mechanism by which CR enhances insulin action, specifically in higher species, is not precisely known. We sought to examine insulin receptor signaling and transcriptional alterations in skeletal muscle of nonhuman primates subjected to CR over a 4-year period. RESEARCH DESIGN AND METHODS At baseline, 32 male adult cynomolgus monkeys (Macaca fascicularis) were randomized to an ad libitum (AL) diet or to 30% CR. Dietary intake, body weight, and insulin sensitivity were obtained at routine intervals over 4 years. At the end of the study, hyperinsulinemic-euglycemic clamps were performed and skeletal muscle (vastus lateralis) was obtained in the basal and insulin-stimulated states for insulin receptor signaling and gene expression profiling. RESULTS CR significantly increased whole-body insulin-mediated glucose disposal compared with AL diet and increased insulin receptor signaling, i.e., insulin receptor substrate (IRS)-1, insulin receptor phosphorylation, and IRS-associated PI 3-kinase activity in skeletal muscle (P < 0.01, P < 0.01, and P < 0.01, respectively). Gene expression for insulin signaling proteins, i.e., IRS-1 and IRS-2, were not increased with CR, although a significant increase in protein abundance was noted. Components of the ubiquitin-proteasome system, i.e., 20S and 19S proteasome subunit abundance and 20S proteasome activity, were significantly decreased by CR. CONCLUSIONS CR increases insulin sensitivity on a whole-body level and enhances insulin receptor signaling in this higher species. CR in cynomolgus monkeys may alter insulin signaling in vivo by modulating protein content of insulin receptor signaling proteins.
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Affiliation(s)
- Zhong Q. Wang
- Division of Nutrition and Chronic Diseases, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana
| | - Z. Elizabeth Floyd
- Division of Nutrition and Chronic Diseases, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana
| | - Jianhua Qin
- Division of Nutrition and Chronic Diseases, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana
| | - Xiaotuan Liu
- Division of Nutrition and Chronic Diseases, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana
| | - Yongmei Yu
- Division of Nutrition and Chronic Diseases, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana
| | - Xian H. Zhang
- Division of Nutrition and Chronic Diseases, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana
| | - Janice D. Wagner
- Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - William T. Cefalu
- Division of Nutrition and Chronic Diseases, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana
- Corresponding author: William T. Cefalu,
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15
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Parathath SR, Mainwaring LA, Fernandez-L A, Campbell DO, Kenney AM. Insulin receptor substrate 1 is an effector of sonic hedgehog mitogenic signaling in cerebellar neural precursors. Development 2008; 135:3291-300. [PMID: 18755774 DOI: 10.1242/dev.022871] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Sonic hedgehog (SHH) and insulin-like growth factor (IGF) signaling are essential for development of many tissues and are implicated in medulloblastoma, the most common solid pediatric malignancy. Cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for specific classes of medulloblastomas, require SHH and IGF signaling for proliferation and survival during development of the cerebellum. We asked whether SHH regulates IGF pathway components in proliferating CGNPs. We report that SHH-treated CGNPs showed increased levels of insulin receptor substrate 1 (IRS1) protein, which was also present in the germinal layer of the developing mouse cerebellum and in mouse SHH-induced medulloblastomas. Previous roles for IRS1, an oncogenic protein that is essential for IGF-mediated proliferation in other cell types, have not been described in SHH-mediated CGNP proliferation. We found that IRS1 overexpression can maintain CGNP proliferation in the absence of SHH. Furthermore, lentivirus-mediated knock down experiments have shown that IRS1 activity is required for CGNP proliferation in slice explants and dissociated cultures. Contrary to traditional models for SHH signaling that focus on gene transcription, SHH stimulation does not regulate Irs1 transcription but rather stabilizes IRS1 protein by interfering with mTOR-dependent IRS1 turnover and possibly affects Irs1 mRNA translation. Thus, we have identified IRS1 as a novel effector of SHH mitogenic signaling that may serve as a future target for medulloblastoma therapies. Our findings also indicate a previously unreported interaction between the SHH and mTOR pathways, and provide an example of a non-classical means for SHH-mediated protein regulation during development.
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Affiliation(s)
- Susana R Parathath
- Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
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16
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Hoehn KL, Hohnen-Behrens C, Cederberg A, Wu LE, Turner N, Yuasa T, Ebina Y, James DE. IRS1-independent defects define major nodes of insulin resistance. Cell Metab 2008; 7:421-33. [PMID: 18460333 PMCID: PMC2443409 DOI: 10.1016/j.cmet.2008.04.005] [Citation(s) in RCA: 237] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/12/2007] [Revised: 02/20/2008] [Accepted: 04/07/2008] [Indexed: 12/16/2022]
Abstract
Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.
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Affiliation(s)
- Kyle L Hoehn
- Diabetes and Obesity Program, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia
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17
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Abstract
First discovered as inhibitors of cytokine signalling, the suppressor of cytokine signalling (SOCS) proteins have appeared, over recent years, as potent repressors of other signalling pathways including the one induced by insulin. SOCS-1 and SOCS-3 have been extensively studied both in vitro and in vivo in the context of insulin action. It has been shown that these two SOCS members are able to inhibit the insulin signalling pathway by three different mechanisms: (1) inhibition of tyrosine phosphorylation of insulin receptor substrate (IRS) proteins because of competition at the docking site on the insulin receptor (IR), (2) induction of the proteasomal degradation of the IRS and (3) inhibition of the IR kinase. A key feature of the SOCS proteins is that they are induced regulators. Indeed, expression of SOCS proteins is virtually absent in basal conditions, but is rapidly and robustly induced in response to several stimuli such as hormones, cytokines and growth factors. A significant correlation between SOCS-3 expression and insulin resistance has been demonstrated in vivo. Interestingly, the level of SOCS-3 expression is strikingly enhanced in insulin-sensitive tissues from both patients and animal models with type 2 diabetes and insulin resistance. While it remains to be established whether the increased expression of SOCS is a cause or a consequence of insulin resistance, a large body of observations supports a role for SOCS proteins in the disease process found in states with insulin resistance.
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Affiliation(s)
- P Lebrun
- Inserm, U145, Faculté de Médecine, Institut de Génétique et Signalisation Moléculaire (IFR50), Université de Nice-Sophia Antipolis, et Laboratoire de Biochimie, CHU, Nice, France
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18
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Prawitt J, Niemeier A, Kassem M, Beisiegel U, Heeren J. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells. Exp Cell Res 2007; 314:814-24. [PMID: 18068701 DOI: 10.1016/j.yexcr.2007.11.011] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2007] [Revised: 11/01/2007] [Accepted: 11/14/2007] [Indexed: 11/28/2022]
Abstract
There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor gamma (PPARgamma) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPARgamma agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA and protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPARgamma-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake.
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Affiliation(s)
- Janne Prawitt
- Department of Biochemistry and Molecular Biology II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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19
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Kang SG, Brown AL, Chung JH. Oxygen Tension Regulates the Stability of Insulin Receptor Substrate-1 (IRS-1) through Caspase-mediated Cleavage. J Biol Chem 2007; 282:6090-7. [PMID: 17179152 DOI: 10.1074/jbc.m610659200] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The insulin and insulin-like growth factor-1 (IGF-1) receptors mediate signaling for energy uptake and growth through insulin receptor substrates (IRSs), which interact with these receptors as well as with downstream effectors. Oxygen is essential not only for ATP production through oxidative phosphorylation but also for many cellular processes, particularly those involved in energy homeostasis. The oxygen tension in vivo is significantly lower than that in the air and can vary widely depending on the tissue as well as on perfusion and oxygen consumption. How oxygen tension affects IRSs and their functions is poorly understood. Our findings indicate that transient hypoxia (1% oxygen) leads to caspase-mediated cleavage of IRS-1 without inducing cell death. The IRS-1 protein level rebounds rapidly upon return to normoxia. Protein tyrosine phosphatases (PTPs) appear to be important for the IRS-1 cleavage because tyrosine phosphorylation of the insulin receptor was decreased in hypoxia and IRS-1 cleavage could be blocked either with H(2)O(2) or with vanadate, each of which inhibits PTPs. Activity of Akt, a downstream effector of insulin and IGF-1 signaling that is known to suppress caspase activation, was suppressed in hypoxia. Overexpression of dominant-negative Akt led to IRS-1 cleavage even in normoxia, and overexpression of constitutively active Akt partially suppressed IRS-1 cleavage in hypoxia, suggesting that hypoxia-mediated suppression of Akt may induce caspase-mediated IRS-1 cleavage. In conclusion, our study elucidates a mechanism by which insulin and IGF-1 signaling can be matched to the oxygen level that is available to support growth and energy metabolism.
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Affiliation(s)
- Sung Gyun Kang
- Laboratory of Biochemical Genetics, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA
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20
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Renström F, Burén J, Svensson M, Eriksson JW. Insulin resistance induced by high glucose and high insulin precedes insulin receptor substrate 1 protein depletion in human adipocytes. Metabolism 2007; 56:190-8. [PMID: 17224332 DOI: 10.1016/j.metabol.2006.09.012] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/28/2006] [Accepted: 09/08/2006] [Indexed: 11/25/2022]
Abstract
The aim of this study was to investigate whether high glucose and/or high insulin produces cellular insulin resistance in human adipocytes and, if so, to evaluate the time course and content of key proteins in the insulin signaling pathway. Subcutaneous fat biopsies were taken from 27 nondiabetic subjects. Insulin action in vitro was studied by measurement of glucose uptake after incubation at a physiologic glucose level (6 mmol/L) for 24 hours or with the last 2, 6, or 24 hours at a high glucose level (20 mmol/L) with or without high insulin (10(4)microU/mL). High glucose alone for 24 hours produced a small but significant impairment (by approximately 20%, P < .05) of insulin's effect to stimulate glucose transport, whereas nonstimulated glucose uptake was left intact. In contrast, the combination of high glucose and high insulin for 6 hours or more reduced basal glucose uptake by approximately 40% (P < .05). In addition, insulin-stimulated glucose uptake capacity was reduced by approximately 40% already after 2 hours (P < .05) and reached a maximal decline (by approximately 50%, P < .05) after a 6-hour culture in high glucose and high insulin. Treatment with high glucose and high insulin in combination for at least 6 hours reduced cellular insulin receptor substrate (IRS)-1, but not IRS-2, protein content by approximately 45% or more (P < .05). Moreover, after 24 hours, the ability of insulin to activate protein kinase B (ie, the phosphorylated protein kinase B [pPKB]-protein kinase B ratio) was decreased by approximately 50% (P < .05). No significant effects were seen on insulin signaling proteins or glucose transporter 4 after a long-term high-glucose culture. Culture with high insulin alone (and low glucose, 6 mmol/L) decreased basal and insulin-stimulated glucose uptake in conformity with the high-glucose/high-insulin setting. However, IRS-1 protein content remained unchanged. We conclude that, in adipocytes from healthy humans, high insulin alone for 2 hours or more decrease glucose uptake capacity. Likewise, high glucose and high insulin in combination for 2 hours or more decrease glucose uptake to the same extent as when cells were cultured with high insulin alone but, in addition, with a diminishment in IRS-1 protein lagging behind. Thus, IRS-1 depletion appears to be a secondary phenomenon in this model of insulin resistance. High glucose alone induces only a minor insulin resistance in human fat cells.
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Affiliation(s)
- Frida Renström
- Department of Public Health and Clinical Medicine, Medicine, Umeå University Hospital, S-901 85 Umeå, Sweden
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21
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Howard JK, Flier JS. Attenuation of leptin and insulin signaling by SOCS proteins. Trends Endocrinol Metab 2006; 17:365-71. [PMID: 17010638 DOI: 10.1016/j.tem.2006.09.007] [Citation(s) in RCA: 257] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/05/2006] [Revised: 09/08/2006] [Accepted: 09/19/2006] [Indexed: 12/13/2022]
Abstract
Leptin and insulin are key hormones involved in the regulation of energy balance and glucose homeostasis. Development of resistance to the action of these hormones, which can occur with age, obesity and inflammation, appears to have a prime role in the pathogenesis of obesity and type 2 diabetes. Specific members of the suppressor of cytokine signaling (SOCS) family of proteins are now thought to have a role in the development of leptin and insulin resistance owing to their ability to inhibit leptin and insulin signaling pathways. In the case of leptin, current evidence suggests that SOCS3 appears to be of particular importance in the development of leptin resistance, whereas the ability to diminish insulin action has been described for several SOCS proteins (SOCS1, SOCS3, SOCS6 and SOCS7).
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Affiliation(s)
- Jane K Howard
- Endocrine Unit, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK
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22
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Sugano T, Yanagita T, Yokoo H, Satoh S, Kobayashi H, Wada A. Enhancement of insulin-induced PI3K/Akt/GSK-3beta and ERK signaling by neuronal nicotinic receptor/PKC-alpha/ERK pathway: up-regulation of IRS-1/-2 mRNA and protein in adrenal chromaffin cells. J Neurochem 2006; 98:20-33. [PMID: 16805793 DOI: 10.1111/j.1471-4159.2006.03846.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
In cultured bovine adrenal chromaffin cells treated with nicotine (10 microm for 24 h), phosphorylation of Akt, glycogen synthase kinase-3beta (GSK-3beta) and extracellular signal-regulated kinase (ERK)1/2 induced by insulin (100 nm for 10 min) was enhanced by approximately 62%, without altering levels of these protein kinases. Nicotine produced time (> 12 h)- and concentration (EC(50) 3.6 and 13 microm)-dependent increases in insulin receptor substrate (IRS)-1 and IRS-2 levels by approximately 125 and 105%, without altering cell surface density of insulin receptors. In these cells, insulin-induced tyrosine phosphorylation of IRS-1/IRS-2 and recruitment of phosphoinositide 3-kinase (PI3K) to IRS-1/IRS-2 were augmented by approximately 63%. The increase in IRS-1/IRS-2 levels induced by nicotine was prevented by nicotinic acetylcholine receptor (nAChR) antagonists, the Ca(2+) chelator 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetrakis-acetoxymethyl ester, cycloheximide or actinomycin D. Nicotine increased IRS-1 and IRS-2 mRNA levels by approximately 57 and approximately 50%, and this was prevented by conventional protein kinase C (cPKC) inhibitor Gö6976, or ERK kinase inhibitors PD98059 and U0126. Nicotine phosphorylated cPKC-alpha, thereby increasing phosphorylation of ERK1/ERK2, as demonstrated by using Gö6976, PD98059 or U0126. Selective activation of cPKC-alpha by thymeleatoxin mimicked these effects of nicotine. Thus, stimulation of nAChRs up-regulated expression of IRS-1/IRS-2 via Ca(2+)-dependent sequential activation of cPKC-alpha and ERK, and enhanced insulin-induced PI3K/Akt/GSK-3beta and ERK signaling pathways.
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Affiliation(s)
- Takashi Sugano
- Department of Pharmacology, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan
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23
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Desbois-Mouthon C, Wendum D, Cadoret A, Rey C, Leneuve P, Blaise A, Housset C, Tronche F, Le Bouc Y, Holzenberger M. Hepatocyte proliferation during liver regeneration is impaired in mice with liver-specific IGF-1R knockout. FASEB J 2006; 20:773-5. [PMID: 16484330 DOI: 10.1096/fj.05-4704fje] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Recent evidence indicates that growth hormone (GH) is involved in liver regeneration. To test whether insulin-like growth factor I (IGF-I) mediates this effect, we studied liver regeneration induced by partial hepatectomy in liver-specific IGF type 1 receptor knockout (LIGFREKO) mice. The absence of IGF-1R caused a significant decrease in hepatocyte proliferation in males (-52%), but not in females, as assessed by Ki67 immunohistochemistry. Cyclin D1 and cyclin A protein levels in the livers of LIGFREKO males were only half those in controls, indicating that cyclin induction during liver regeneration is dependent on IGF-1R signaling. Analyzing the signaling cascade initiated by IGF-1R, we observed a lack of IRS-1 induction in LIGFREKO livers. In contrast, the induction of IRS-2 synthesis was similar in LIGFREKO and control groups, suggesting the existence of differential regulation of IRS synthesis during liver regeneration. Regenerating livers from LIGFREKO animals also showed significantly less activated ERKs than controls. Our findings demonstrate that IGF-1R makes a significant contribution to liver regeneration. Using the LIGFREKO model, we provide new evidence that IGF-1R/IRS-1/ERK signaling may be the intracellular pathway controlling the cell cycle via cyclin D1 and cyclin A in the regenerating liver.
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24
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Johnson DR, O'Connor JC, Satpathy A, Freund GG. Cytokines in type 2 diabetes. VITAMINS AND HORMONES 2006; 74:405-41. [PMID: 17027525 DOI: 10.1016/s0083-6729(06)74017-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Affiliation(s)
- Daniel R Johnson
- Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801, USA
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25
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Bloch-Damti A, Bashan N. Proposed mechanisms for the induction of insulin resistance by oxidative stress. Antioxid Redox Signal 2005; 7:1553-67. [PMID: 16356119 DOI: 10.1089/ars.2005.7.1553] [Citation(s) in RCA: 276] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
In diabetes (type 1 and type 2), increased flux of free fatty acids and glucose is associated with increased mitochondrial reactive oxygen species (ROS) production and, as a consequence, increased oxidative stress. ROS have been shown to activate various cellular stress-sensitive pathways, which can interfere with cellular signaling pathways. Exposure of different cell lines to micromolar concentrations of hydrogen peroxide leads to the activation of stress kinases such as c-Jun N-terminal kinase, p38, I kappaB kinase, and extracellular receptor kinase 1/2. This activation is accompanied by a down-regulation of the cellular response to insulin, leading to a reduced ability of insulin to promote glucose uptake, and glycogen and protein synthesis. The mechanisms leading to this down-regulation in oxidized cells are complicated, involving increased serine/threonine phosphorylation of insulin receptor substrate-1 (IRS1), impaired insulin-stimulated redistribution of IRS1 and phosphatidylinositol-kinase between cytosol and low-density microsomal fraction, followed by a reduced protein kinase-B phosphorylation and GLUT4 translocation to the plasma membrane. In addition, prolonged exposure to ROS affects transcription of glucose transporters: whereas the level of GLUT1 is increased, GLUT4 level is reduced. As can be expected, administration of antioxidants such as lipoic acid in oxidized cells, in animal models of diabetes, and in type 2 diabetes shows improved insulin sensitivity. Thus, oxidative stress is presently accepted as a likely causative factor in the development of insulin resistance.
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Affiliation(s)
- Asnat Bloch-Damti
- Department of Clinical Biochemistry, Soroka Medical Center, Ben-Gurion University of the Negev, Beer-Sheva, Israel
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26
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Banks AS, Li J, McKeag L, Hribal ML, Kashiwada M, Accili D, Rothman PB. Deletion of SOCS7 leads to enhanced insulin action and enlarged islets of Langerhans. J Clin Invest 2005; 115:2462-71. [PMID: 16127460 PMCID: PMC1190369 DOI: 10.1172/jci23853] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2004] [Accepted: 06/14/2005] [Indexed: 12/13/2022] Open
Abstract
NIDDM is characterized by progressive insulin resistance and the failure of insulin-producing pancreatic beta cells to compensate for this resistance. Hyperinsulinemia, inflammation, and prolonged activation of the insulin receptor (INSR) have been shown to induce insulin resistance by decreasing INSR substrate (IRS) protein levels. Here we describe a role for SOCS7 in regulating insulin signaling. Socs7-deficient mice exhibited lower glucose levels and prolonged hypoglycemia during an insulin tolerance test and increased glucose clearance in a glucose tolerance test. Six-month-old Socs7-deficient mice exhibited increased growth of pancreatic islets with mildly increased fasting insulin levels and hypoglycemia. These defects correlated with increased IRS protein levels and enhanced insulin action in cells lacking SOCS7. Additionally, SOCS7 associated with the INSR and IRS1--molecules that are essential for normal regulation of insulin action. These data suggest that SOCS7 is a potent regulator of glucose homeostasis and insulin signaling.
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Affiliation(s)
- Alexander S Banks
- Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
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27
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Renström F, Burén J, Eriksson JW. Insulin receptor substrates-1 and -2 are both depleted but via different mechanisms after down-regulation of glucose transport in rat adipocytes. Endocrinology 2005; 146:3044-51. [PMID: 15845625 DOI: 10.1210/en.2004-1675] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Alterations in muscle and adipose tissue insulin receptor substrate (IRS)-1 and IRS-2 are associated with, and commonly believed to contribute to, development of insulin resistance. In this study, we investigated the mechanisms behind previously observed reductions in IRS levels due to high concentrations of glucose and insulin and their significance in the impairment of glucose uptake capacity in primary rat adipocytes. Semiquantitative RT-PCR analysis showed that insulin (10(4) microU/ml) alone or in combination with glucose (15 mm) markedly suppressed IRS-2 gene expression, whereas IRS-1 mRNA was unaffected by the culture conditions. The negative effect of a high glucose/high insulin setting on IRS-1 protein level was still exerted when protein synthesis was inhibited with cycloheximide. Impairment of glucose uptake capacity after treatment with high glucose and insulin was most pronounced after 3 h, whereas IRS-1 and IRS-2 protein levels were unaffected up to 6 h but were reduced after 16 h. Moreover, impaired glucose uptake capacity could only partially be reversed by subsequent incubation at physiological conditions. These novel results suggest that: 1) in a high glucose/high insulin setting depletion of IRS-1 and IRS-2 protein, respectively, occurs via different mechanisms, and IRS-2 gene expression is suppressed, whereas IRS-1 depletion is due to posttranslational mechanisms; 2) IRS-1 and IRS-2 protein depletion is a secondary event in the development of insulin resistance in this model of hyperglycemia/hyperinsulinemia; and 3) depletion of cellular IRS in adipose tissue may be a consequence rather than a cause of insulin resistance and hyperinsulinemia in type 2 diabetes.
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Affiliation(s)
- Frida Renström
- Department of Medicine, Umeå University Hospital, SE-901 85 Umeå, Sweden
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28
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Kairouz R, Parmar J, Lyons RJ, Swarbrick A, Musgrove EA, Daly RJ. Hormonal regulation of the Grb14 signal modulator and its role in cell cycle progression of MCF-7 human breast cancer cells. J Cell Physiol 2005; 203:85-93. [PMID: 15372466 DOI: 10.1002/jcp.20199] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Growth factor receptor bound (Grb)14 is a member of the Grb7 family of src homology (SH)2 domain-containing proteins. These proteins perform both adaptor and modulatory roles in receptor tyrosine kinase (RTK) signaling, although their regulation is poorly understood. In this study, a positive correlation between Grb14 protein expression and ER alpha status in breast cancer cell lines led us to investigate regulation of Grb14 by estradiol and insulin, which synergize in the regulation of breast cancer cell proliferation. In MCF-7 cells maintained in charcoal-stripped serum, Grb14 expression was downregulated by estradiol and increased by the pure anti-estrogen ICI 182780. Under serum-free conditions, insulin enhanced Grb14 expression but this effect was repressed by estradiol when both hormones were used in combination. Using a system in which c-Myc induction drives cell cycle progression independently of estradiol, we demonstrated that Grb14 regulation was specific to estradiol treatment. Finally, we demonstrated a novel functional role for Grb14 whereby its overexpression inhibited not only insulin- but also estrogen-induced cell cycle progression. This was associated with decreased extracellular signal-regulated kinase (Erk)1/2 activation in insulin-stimulated Grb14-overexpressing cells. These data represent the first demonstration of regulation of Grb14 expression levels in response to hormonal stimuli, and are consistent with its role as a repressor of insulin signaling where it is induced as a negative feedback mechanism. A role for Grb14 is also shown in estrogen/insulin crosstalk since estradiol blocks the insulin-induced induction of this protein.
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Affiliation(s)
- Rania Kairouz
- Cancer Research Program, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, New South Wales, Australia
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29
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Björnholm M, Zierath JR. Insulin signal transduction in human skeletal muscle: identifying the defects in Type II diabetes. Biochem Soc Trans 2005; 33:354-7. [PMID: 15787605 DOI: 10.1042/bst0330354] [Citation(s) in RCA: 143] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Type II diabetes is characterized by defects in insulin action on peripheral tissues, such as skeletal muscle, adipose tissue and liver and pancreatic β-cell defects. Since the skeletal muscle accounts for approx. 75% of whole body insulin-stimulated glucose uptake, defects in this tissue play a major role in the impaired glucose homoeostasis in Type II diabetic patients. Thus identifying defective steps in this process may reveal attractive targets for drug development to combat insulin resistance and Type II diabetes. This review will describe the effects of insulin on glucose transport and other metabolic events in skeletal muscle that are mediated by intracellular signalling cascades. Evidence for impaired activation of the insulin receptor signalling cascade and defective glucose transporter 4 translocation in the skeletal muscle from Type II diabetic patients will be presented. Through the identification of the intracellular defects in insulin action that control glucose homoeostasis, a better understanding of the disease pathogenesis can be gained and strategies for intervention may be developed.
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Affiliation(s)
- M Björnholm
- Department of Surgical Sciences, Integrative Physiology, Karolinska Institutet, Stockholm, Sweden
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30
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Usui I, Imamura T, Huang J, Satoh H, Shenoy SK, Lefkowitz RJ, Hupfeld CJ, Olefsky JM. beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1. Mol Cell Biol 2004; 24:8929-37. [PMID: 15456867 PMCID: PMC517874 DOI: 10.1128/mcb.24.20.8929-8937.2004] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.
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Affiliation(s)
- Isao Usui
- Department of Medicine (0673), University of California, San Diego, Stein Bldg, Room 210, 9500 Gilman Dr., La Jolla, CA 92093-0673, USA
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31
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Shi H, Tzameli I, Bjørbaek C, Flier JS. Suppressor of cytokine signaling 3 is a physiological regulator of adipocyte insulin signaling. J Biol Chem 2004; 279:34733-40. [PMID: 15181014 DOI: 10.1074/jbc.m403886200] [Citation(s) in RCA: 177] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Many proinflammatory cytokines and hormones have been demonstrated to be involved in insulin resistance. However, the molecular mechanisms whereby these cytokines and hormones inhibit insulin signaling are not completely understood. We observed that several cytokines and hormones that induce insulin resistance also stimulate SOCS3 expression in 3T3-L1 adipocytes and that SOCS3 mRNA is increased in adipose tissue of obese/diabetic mice. We then hypothesized that SOCS3 may mediate cytokine- and hormone-induced insulin resistance. By using SOCS3-deficient adipocytes differentiated from mouse embryonic fibroblasts, we found that SOCS3 deficiency increases insulin-stimulated IRS1 and IRS2 phosphorylation, IRS-associated phosphatidylinositol 3-kinase activity, and insulin-stimulated glucose uptake. Moreover, lack of SOCS3 substantially limits the inhibitory effects of tumor necrosis factor-alpha to suppress IRS1 and IRS2 tyrosine phosphorylation, phosphatidylinositol 3-kinase activity, and glucose uptake in adipocytes. The ameliorated insulin signaling in SOCS3-deficient adipocytes is mainly due to the suppression of tumor necrosis factor-alpha-induced IRS1 and IRS2 protein degradation. Therefore, our data suggest that endogenous SOCS3 expression is a key determinant of basal insulin signaling and is an important molecular mediator of cytokine-induced insulin resistance in adipocytes. We conclude that SOCS3 plays an important role in mediating insulin resistance and may be an excellent target for therapeutic intervention in insulin resistance and type II diabetes.
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Affiliation(s)
- Hang Shi
- Division of Endocrinology, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA
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32
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Castro FCP, Delgado EF, Bezerra RMN, Lanna DPD. Effects of growth hormone on insulin signal transduction in rat adipose tissue maintained in vitro. Endocr Res 2004; 30:225-38. [PMID: 15473132 DOI: 10.1081/erc-120039578] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Growth hormone treatment (GH) decreases adipose tissue sensitivity to insulin. However, the exact molecular mechanism(s) involved remains unclear. In the present study, we have evaluated the chronic effects of GH on adipose tissue explants cultured in a defined media. The objective was to determine the effects of GH treatment for 24 and 48 hours on the early steps of the insulin signal transduction, including IRS-3. The 24-hour culture media contained no hormones or 100 ng/ml GH. The 48-hour culture media contained insulin and dexamethasone supplemented with or without 100 ng/ml of GH. Results demonstrated a reduction in the cellular concentration of IRS-1 by around 30% when adipose tissue was chronically treated with growth hormone for either 24 or 48 hours. IRS-3 protein levels were also decreased by 15% after the 24-hour treatment, and by 27% after culture with GH for 48 hours in the presence of insulin and dexamethasone. PI 3-kinase concentrations were also reduced by GH in both experiments by around 25%. At the end of the 24-hour culture with GH adipose explants were stimulated with insulin in a short-term incubation, after which phosphorylation and association of the IRSs with PI 3-kinase were evaluated. After the insulin stimulus, the association of PI 3-kinase with IRS-1 and IRS-3 were decreased in explants chronically cultured with GH by 44 and 28%, respectively. After this short-term insulin stimulus, the IRS-3 phosphorylation was also lowered in GH-treated explants. The results with chronic cultures of adipose presented here are consistent with similar changes in IRS-1 and IRS-2 concentration and phosphorylation observed for liver and muscle after long-term (3-5 days) in vivo treatment with GH. The data suggest that chronic GH treatment alters the early steps of the insulin signal transduction pathway, and may explain the changes in adipose tissue sensitivity to insulin.
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Affiliation(s)
- Fernanda C P Castro
- Departamento de Zootecnia, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, São Paulo, Brazil
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33
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Cruz M, Velasco E, Kumate J. Degradation of pro-insulin-receptor proteins by proteasomes. Arch Med Res 2004; 35:18-23. [PMID: 15036795 DOI: 10.1016/j.arcmed.2003.08.008] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2002] [Accepted: 08/29/2003] [Indexed: 11/21/2022]
Abstract
BACKGROUND Type-2 diabetes is characterized by hyperinsulinemia, peripheral insulin resistance, and diminished tyrosine phosphorylation activity. It has been recently shown that proteasomes are implicated in the degradation of the insulin receptor substrate-1 (IRS-1) but not in that of the insulin receptor (IR). However, it is unknown whether proteasomes are involved in pro-IR degradation. METHODS We used CHO-IR and the 3T3-L1 cells treated with insulin at different concentrations and compared the proteasome activity of IRS-1, IR, and pro-IR degradation either in presence or in absence of lactacystin. RESULTS A total of 100 nM of insulin allowed degradation of IRS-1 after 6 h of incubation. At 1,000 nM of insulin, pro-IR degradation began at 1 h of incubation, similar to IRS-1 degradation. Surprisingly, at a higher concentration (10 microM) of insulin, a drastic decrease of proteins was observed from the first minute of incubation. This activity was blocked by lactacystin, a specific proteasome inhibitor. CONCLUSIONS According to these results, we propose that pro-IR is degraded by proteasomes.
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Affiliation(s)
- Miguel Cruz
- Unidad de Investigación Medica en Bioquímica, Hospital de Especialidades, Centro Medico Nacional Siglo XXI (CMNSXXI), Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico.
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Keller SR, Lienhard GE. Insulin signalling: the role of insulin receptor substrate 1. Trends Cell Biol 2004; 4:115-9. [PMID: 14731733 DOI: 10.1016/0962-8924(94)90065-5] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The insulin receptor is a ligand-activated tyrosine kinase that phosphorylates its major substrate protein, insulin receptor substrate 1 (IRS1), at multiple sites. Tyrosine-phosphorylated IRS1 then serves as a docking/effector protein for at least four Src homology 2 (SH2)-domain proteins involved in signal transduction. This initial step in signalling distinguishes the insulin receptor from other receptor tyrosine kinases, which directly bind several SH2-domain proteins, and establishes IRS1 as a founding member of a group of proteins whose function is to link activated tyrosine kinases to SH2-domain proteins.
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Affiliation(s)
- S R Keller
- Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA
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35
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Abstract
Insulin is a key hormone regulating the control of metabolism and the maintenance of normoglycaemia and normolipidaemia. Insulin acts by binding to its cell surface receptor, thus activating the receptor's intrinsic tyrosine kinase activity, resulting in receptor autophosphorylation and phosphorylation of several substrates. Tyrosine phosphorylated residues on the receptor itself and on subsequently bound receptor substrates provide docking sites for downstream signalling molecules, including adapters, protein serine/threonine kinases, phosphoinositide kinases and exchange factors. Collectively, those molecules orchestrate the numerous insulin-mediated physiological responses. A clear picture is emerging of the way in which insulin elicits several intracellular signalling pathways to mediate its physiologic functions. A further challenge, being pursued by several laboratories, is to understand the molecular mechanisms that underlie insulin action at the peripheral level, deregulation of which ultimately leads to hyperglycaemia and Type 2 diabetes. We review how circulating factors such as insulin itself, TNF-alpha, interleukins, fatty acids and glycation products influence insulin action through insulin signalling molecules themselves or through other pathways ultimately impinging on the insulin-signalling pathway. Understanding how the mechanism by which molecular insulin action is modulated by these factors will potentially provide new targets for pharmacological agents, to enable the control of altered glucose and lipid metabolism and diabetes.
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Affiliation(s)
- L Pirola
- INSERM Unit 145, Faculty of Medicine, Nice, France
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36
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Zhang H, Hoff H, Sell C. Downregulation of IRS-1 protein in thapsigargin-treated human prostate epithelial cells. Exp Cell Res 2003; 289:352-8. [PMID: 14499636 DOI: 10.1016/s0014-4827(03)00286-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Thapsigargin treatment of cultured cells leads to an increase in the intracellular calcium concentration, activation of calpain, and, in some cell types, apoptosis. Using a human prostate epithelial cell line that undergoes apoptosis in the presence of thapsigargin, we find decreased levels of IRS-1 protein levels during apoptosis. Inhibition of calpain prevents this decrease in IRS-1 protein; however, inhibitors of caspases or the proteasome are ineffective in maintaining IRS-1 levels. In terms of IGF-I-related second messenger proteins, the effect of thapsigargin is specific for IRS-1 since the protein levels of IGF-I receptor beta-subunit, Akt, Erk, and Shc are not affected. In addition to preventing the reduction in IRS-1, treatment of cells with calpain inhibitor II prevents apoptosis in response to thapsigargin. Finally, IRS-1 and calpain can be identified in protein complexes isolated using IRS-1-specific antibodies, indicating that calpain can associate with either IRS-1 or one of the proteins present in protein complexes that contain IRS-1. In total, these results suggest that IRS-1 may be targeted for degradation by calpain during apoptosis.
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Affiliation(s)
- Hong Zhang
- Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
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Morelli C, Garofalo C, Bartucci M, Surmacz E. Estrogen receptor-alpha regulates the degradation of insulin receptor substrates 1 and 2 in breast cancer cells. Oncogene 2003; 22:4007-16. [PMID: 12821935 DOI: 10.1038/sj.onc.1206436] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
In breast cancer cells, 17-beta-estradiol (E2) upregulates the expression of insulin receptor substrate 1 (IRS-1), a molecule transmitting insulin-like growth factor-I (IGF-I) signals through the PI-3K/Akt survival pathways. The stimulation of IRS-1 by E2 has been documented on the transcriptional level. Here we studied whether the expression of estrogen receptor (ER)-alpha affects IRS molecules post-transcriptionally. We used ER-alpha-negative MDA-MB-231 breast cancer cells and MDA-MB-231 cells with re-expressed ER-alpha. In MDA-MB-231 cells cultured under serum-free conditions, IRS-1 and IRS-2 were degraded through the 26S proteasome and calpain pathways. Re-expression of ER-alpha in MDA-MB-231 cells correlated with enhanced stability of IRS molecules. This effect coincided with significantly reduced ubiquitination of IRS-1 and IRS-2, but did not involve increased IRS-1 and IRS-2 transcription. The interference of ER-alpha with IRS-1 and IRS-2 turnover could rely on the competition for common degradation pathways, as in MDA-MB-231/ER cells, ER-alpha processing was blocked by proteasome and calpain inhibitors. Notably, a fraction of the cytosolic ER-alpha colocalized and coprecipitated with IRS-1 and IRS-2, indicating a possible common destination for these proteins. The stabilization of IRS-1 in MDA-MB-231/ER cells was paralleled by the upregulation of the IRS-1/Akt/GSK-3 pathway and improved survival in the presence of IGF-I, whereas IRS-2 was not involved in IGF-I signaling.
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Affiliation(s)
- Catia Morelli
- Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA
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38
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Abstract
Tyrosine dephosphorylation, serine phosphorylation, and proteasomal degradation of insulin receptor substrates (IRSs) are implicated in the negative regulation of insulin action. Here we show that simultaneous inhibition of IRS-1 tyrosine dephosphorylation and proteasomal degradation synergistically augments insulin-responsive glucose uptake. L6 skeletal muscle cells (L6 cells) were treated with inhibitors of protein-tyrosine phosphatases, proteasomal degradation, and mammalian target of rapamycin (mTOR), and the effects of insulin on glucose uptake, IRS-1 tyrosine phosphorylation, phosphatidylinositol (PI) 3-kinase activity, and IRS-1 mass were examined. Pretreatment of L6 cells with sodium orthovanadate (Na(3)VO(4)) plus the mTOR inhibitor rapamycin caused a 5-fold increase in insulin-responsive glucose uptake at 2 hours when compared to insulin alone. Evaluation of IRS-1 associated PI 3-kinase activity, IRS-1-associated p85 mass, and IRS-1 tyrosine phosphorylation showed that 2 hours after insulin addition they were reduced by 70% from maximal activity. Likewise, IRS-1 mass was reduced by 50%. When L6 cells were pretreated with Na(3)VO(4) plus the proteasome inhibitor MG-132 or the mTOR inhibitor rapamycin prior to insulin addition, IRS-1 mass loss as well as IRS-1/PI-3 kinase complex decay was blocked at 2 hours and PI 3-kinase activity was increased 2.5-fold and 4-fold, respectively, over insulin alone. Finally, treatment of L6 cells with subtherapeutic amounts of vanadyl sulfate and rapamycin induced a synergistic 3-fold increase in insulin-induced glucose uptake at 2 hours. These findings indicate that vanadium and rapamycin synergize to enhance glucose uptake by preventing IRS-1 mass loss and IRS-1/PI 3-kinase complex decay and may offer a new approach to enhance glucose transport in diabetes.
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Affiliation(s)
- Jason C O'Connor
- Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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39
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Pirola L, Bonnafous S, Johnston AM, Chaussade C, Portis F, Van Obberghen E. Phosphoinositide 3-kinase-mediated reduction of insulin receptor substrate-1/2 protein expression via different mechanisms contributes to the insulin-induced desensitization of its signaling pathways in L6 muscle cells. J Biol Chem 2003; 278:15641-51. [PMID: 12594228 DOI: 10.1074/jbc.m208984200] [Citation(s) in RCA: 89] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to balance peripheral insulin resistance. To gain insight into the deleterious effects of hyperinsulinemia on skeletal muscle, we studied the consequences of prolonged insulin treatment of L6 myoblasts on insulin-dependent signaling pathways. A 24-h long insulin treatment desensitized the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) and p42/p44 MAPK pathways toward a second stimulation with insulin or insulin-like growth factor-1 and led to decreased insulin-induced glucose uptake. Desensitization was correlated to a reduction in insulin receptor substrate (IRS)-1 and IRS-2 protein levels, which was reversed by the PI3K inhibitor LY294002. Co-treatment of cells with insulin and LY294002, while reducing total IRS-1 phosphorylation, increased its phosphotyrosine content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK inhibitors did not block insulin-induced reduction of IRS-1, suggesting that the PI3K serine-kinase activity causes IRS-1 serine phosphorylation and its commitment to proteasomal degradation. Contrarily, insulin-induced IRS-2 down-regulation occurred via a PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002 rescued the responsiveness of PKB and MAPK toward acute insulin stimulation. Conversely, adenoviral-driven expression of constitutively active PI3K induced an insulin-independent reduction in IRS-1/2 protein levels. IRS-2 appears to be the chief molecule responsible for MAPK and PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA interference severely impaired activation of both kinases. In summary, (i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating it while a PI3K/mTOR pathway controls insulin-induced reduction of IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking the insulin receptor to activation of PKB and MAPK, (iii) the mechanism of IRS-1/2 down-regulation is different in L6 cells compared with 3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via different PI3K-mediated mechanisms contributes to the development of an insulin-resistant state in L6 myoblasts.
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Affiliation(s)
- Luciano Pirola
- INSERM U145, IFR50, Faculté de Médecine, 06107 Nice Cedex 2, France
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Potashnik R, Bloch-Damti A, Bashan N, Rudich A. IRS1 degradation and increased serine phosphorylation cannot predict the degree of metabolic insulin resistance induced by oxidative stress. Diabetologia 2003; 46:639-48. [PMID: 12750770 DOI: 10.1007/s00125-003-1097-5] [Citation(s) in RCA: 89] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/08/2002] [Revised: 12/20/2002] [Indexed: 11/30/2022]
Abstract
AIM/HYPOTHESIS Oxidative stress was shown to selectively induce impaired metabolic response to insulin, raising the possible involvement of alterations in Insulin-Receptor-Substrate (IRS) proteins. This study was conducted to assess whether oxidative stress induced IRS protein degradation and enhanced serine phosphorylation, and to assess their functional importance. METHODS 3T3-L1 adipocytes and rat hepatoma cells (FAO) were exposed to micro-molar H(2)O(2) by adding glucose oxidase to the culture medium, and IRS1 content, its serine phosphorylation and downstream metabolic insulin effects were measured. RESULTS Cells exposed to oxidative stress exhibited decreased IRS1 (but not IRS2) content, and increased serine phosphorylation of both proteins. Total protein ubiquitination was increased in oxidized cells, but not in cells exposed to prolonged insulin treatment. Yet, lactacystin and MG132, two unrelated proteasome inhibitors, prevented IRS1 degradation induced by prolonged insulin but not by oxidative stress. The PI 3-kinase inhibitor LY294002 and the mTOR inhibitor rapamycin, but not the MEK1 inhibitor PD98059, could prevent IRS1 changes in oxidized cells. Rapamycin, which protected against IRS1 degradation and serine phosphorylation was not associated with improved response to acute insulin stimulation. Moreover, the antioxidant alpha lipoic acid, while protecting against oxidative stress-induced insulin resistance in 3T3-L1 adipocytes, could not prevent IRS1 degradation and serine phosphorylation. CONCLUSION/INTERPRETATION Oxidative stress induces serine phosphorylation of IRS1 and increases its degradation by a proteasome-independent pathway; yet, these changes do not correlate with the induction of impaired metabolic response to insulin.
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Affiliation(s)
- R Potashnik
- Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel
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41
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Greene MW, Sakaue H, Wang L, Alessi DR, Roth RA. Modulation of insulin-stimulated degradation of human insulin receptor substrate-1 by Serine 312 phosphorylation. J Biol Chem 2003; 278:8199-211. [PMID: 12510059 DOI: 10.1074/jbc.m209153200] [Citation(s) in RCA: 162] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Ser/Thr phosphorylation of insulin receptor substrate-1 (IRS-1) is a negative regulator of insulin signaling. One potential mechanism for this is that Ser/Thr phosphorylation decreases the ability of IRS-1 to be tyrosine-phosphorylated by the insulin receptor. An additional mechanism for modulating insulin signaling is via the down-regulation of IRS-1 protein levels. Insulin-induced degradation of IRS-1 has been well documented, both in cells as well as in patients with diabetes. Ser/Thr phosphorylation of IRS-1 correlates with IRS-1 degradation, yet the details of how this occurs are still unknown. In the present study we have examined the potential role of different signaling cascades in the insulin-induced degradation of IRS-1. First, we found that inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin block the degradation. Second, knockout cells lacking one of the key effectors of this cascade, the phosphoinositide-dependent kinase-1, were found to be deficient in the insulin-stimulated degradation of IRS-1. Conversely, overexpression of this enzyme potentiated insulin-stimulated IRS-1 degradation. Third, concurrent with the decrease in IRS-1 degradation, the inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin also blocked the insulin-stimulated increase in Ser(312) phosphorylation. Most important, an IRS-1 mutant in which Ser(312) was changed to alanine was found to be resistant to insulin-stimulated IRS-1 degradation. Finally, an inhibitor of c-Jun N-terminal kinase, SP600125, at 10 microm did not block IRS-1 degradation and IRS-1 Ser(312) phosphorylation yet completely blocked insulin-stimulated c-Jun phosphorylation. Further, insulin-stimulated c-Jun phosphorylation was not blocked by inhibitors of the phosphatidylinositol 3-kinase and mammalian target of rapamycin, indicating that c-Jun N-terminal kinase is unlikely to be the kinase phosphorylating IRS-1 Ser(312) in response to insulin. In summary, our results indicate that the insulin-stimulated degradation of IRS-1 via the phosphatidylinositol 3-kinase pathway is in part dependent upon the Ser(312) phosphorylation of IRS-1.
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Affiliation(s)
- Michael W Greene
- Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305, USA
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42
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Dalle S, Imamura T, Rose DW, Worrall DS, Ugi S, Hupfeld CJ, Olefsky JM. Insulin induces heterologous desensitization of G-protein-coupled receptor and insulin-like growth factor I signaling by downregulating beta-arrestin-1. Mol Cell Biol 2002; 22:6272-85. [PMID: 12167719 PMCID: PMC134007 DOI: 10.1128/mcb.22.17.6272-6285.2002] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
beta-Arrestin-1 mediates agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCRs) and is also essential for GPCR mitogenic signaling. In addition, insulin-like growth factor I receptor (IGF-IR) endocytosis is facilitated by beta-arrestin-1, and internalization is necessary for IGF-I-stimulated mitogen-activated protein (MAP) kinase activation. Here, we report that treatment of cells for 12 h with insulin (100 ng/ml) induces an approximately 50% decrease in cellular beta-arrestin-1 content due to ubiquitination of beta-arrestin-1 and proteosome-mediated degradation. This insulin-induced decrease in beta-arrestin-1 content was blocked by inhibition of phosphatidylinositol-3 kinase (PI-3 kinase) and MEK with wortmannin and PD98059, respectively. We also found a marked decrease in the association of beta-arrestin-1 with the IGF-IR and a 55% inhibition of IGF-I-stimulated MAP kinase phosphorylation. In insulin-treated, beta-arrestin-1-downregulated cells, there was complete inhibition of lysophosphatidic acid (LPA) or isoproterenol (ISO)-stimulated MAP kinase phosphorylation. This was associated with a decrease in beta-arrestin-1 association with the beta2-AR as well as a decrease in beta-arrestin-1-Src and Src-beta2-AR association. Ectopic expression of wild-type beta-arrestin-1 in insulin-treated cells in which endogenous beta-arrestin-1 had been downregulated rescued IGF-I- and LPA-stimulated MAP kinase phosphorylation. In conclusion, we found the following. (i) Chronic insulin treatment leads to enhanced beta-arrestin-1 degradation. (ii) This downregulation of endogenous beta-arrestin-1 is associated with decreased IGF-I-, LPA-, and ISO-mediated MAP kinase signaling, which can be rescued by ectopic expression of wild-type beta-arrestin-1. (iii) Finally, these results describe a novel mechanism for heterologous desensitization, whereby insulin treatment can impair GPCR signaling, and highlight the importance of beta-arrestin-1 as a target molecule for this desensitization mechanism.
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Affiliation(s)
- Stéphane Dalle
- Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673, USA
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Chan CMW, Martin LA, Johnston SRD, Ali S, Dowsett M. Molecular changes associated with the acquisition of oestrogen hypersensitivity in MCF-7 breast cancer cells on long-term oestrogen deprivation. J Steroid Biochem Mol Biol 2002; 81:333-41. [PMID: 12361723 DOI: 10.1016/s0960-0760(02)00074-2] [Citation(s) in RCA: 98] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The growth dependence of many breast cancers on oestrogen has been exploited therapeutically by oestrogen deprivation, but almost all patients eventually develop resistance largely by unknown mechanisms. Wild-type (WT) MCF-7 cells were cultured in oestrogen-deficient medium for 90 weeks in order to establish a long-term oestrogen-deprived MCF-7 (LTED) which eventually became independent of exogenous oestrogen for growth. After 15 weeks of quiescence (LTED-Q), basal growth rate increased in parallel with increasing oestrogen sensitivity. While 10(-9)M oestradiol (E2) maximally stimulated WT growth, the hypersensitive LTED (LTED-H) were maximally growth stimulated by 10(-13)M E2. By week 50, hypersensitivity was apparently lost and the cells became oestrogen independent (LTED-I), although the pure antioestrogen ICI182780 still inhibited cell growth and reversed the inhibitory effect of 10(-9)M E2 at 10(-12) to 10(-7)M. Tamoxifen (10(-7) to 10(-6)M) had a partial agonist effect on WT, but had no stimulatory effect on LTED. Whilst LTED cells have a low progesterone receptor (PgR) expression in all phases, oestrogen receptor (ER) a expression was, on average, elevated five- and seven-fold in LTED-H and LTED-I, respectively, and serine118 was phosphorylated. ERbeta expression was up-regulated and the levels of insulin receptor substrate 1 (IRS-1) remained low throughout all phases. The levels of RIP140mRNA appeared to decrease to approximately 50% of the WT message in LTED-Q and remained constant into the hypersensitive phase. No significant changes were observed in the expression of SUG-1, TIF-1 and SMRT in LTED. The overall changes in nuclear receptor interacting proteins do not appear to be involved in the hypersensitivity. Thus, the resistance of these human breast cancer cells to oestrogen-deprivation appears to be due to acquired hypersensitivity which may be explained in part by increased levels of and phosphorylated ERalpha.
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MESH Headings
- ATPases Associated with Diverse Cellular Activities
- Adaptor Proteins, Signal Transducing
- Blotting, Northern
- Blotting, Western
- Breast Neoplasms/drug therapy
- Breast Neoplasms/metabolism
- Carrier Proteins/genetics
- Carrier Proteins/metabolism
- Cell Division/drug effects
- DNA-Binding Proteins/genetics
- DNA-Binding Proteins/metabolism
- Drug Resistance, Neoplasm
- Estradiol/analogs & derivatives
- Estradiol/pharmacology
- Estrogen Antagonists/pharmacology
- Estrogen Receptor Modulators/pharmacology
- Estrogen Receptor alpha
- Estrogen Receptor beta
- Estrogens/deficiency
- Estrogens/pharmacology
- Female
- Fulvestrant
- Gene Expression Regulation, Neoplastic
- Humans
- Insulin Receptor Substrate Proteins
- Intracellular Signaling Peptides and Proteins
- LIM Domain Proteins
- Nuclear Proteins/genetics
- Nuclear Proteins/metabolism
- Nuclear Receptor Co-Repressor 2
- Nuclear Receptor Interacting Protein 1
- Phosphoproteins/metabolism
- Phosphorylation/drug effects
- Proteasome Endopeptidase Complex
- Protozoan Proteins
- RNA, Messenger/metabolism
- Receptors, Estrogen/metabolism
- Receptors, Progesterone/metabolism
- Repressor Proteins/genetics
- Repressor Proteins/metabolism
- Tamoxifen/pharmacology
- Transcription Factors
- Tumor Cells, Cultured/drug effects
- Tumor Cells, Cultured/metabolism
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Affiliation(s)
- Christina M W Chan
- Department of Academic Biochemistry, Royal Marsden Hospital, Fulham Road, London SW3 6JJ, UK
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44
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Berg CE, Lavan BE, Rondinone CM. Rapamycin partially prevents insulin resistance induced by chronic insulin treatment. Biochem Biophys Res Commun 2002; 293:1021-7. [PMID: 12051762 DOI: 10.1016/s0006-291x(02)00333-9] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Chronic insulin exposure induces serine/threonine phosphorylation and degradation of IRS-1 through a rapamycin-sensitive pathway, which results in a down-regulation of insulin action. In this study, to investigate whether rapamycin (an mTOR inhibitor) could prevent insulin resistance induced by hyperinsulinemia, 3T3-L1 adipocytes were incubated chronically in the presence of insulin with or without the addition of rapamycin. Subsequently, the cells were washed and re-stimulated acutely with insulin. Chronic insulin stimulation caused a reduction of GLUT-4 and IRS-1 proteins with a correlated decrease in acute insulin-induced PKB and MAPK phosphorylations as well as a reduction in insulin-stimulated glucose transport. Rapamycin prevented the reduction of IRS-1 protein levels and insulin-induced PKB Ser-473 phosphorylation with a partial normalization of insulin-induced glucose transport. In contrast, rapamycin had no effect on the decrease in insulin-induced MAPK phosphorylation or GLUT-4 protein levels. These results suggest that chronic insulin exposure leads to a down-regulation of PKB and MAPK pathways through different mechanisms in adipocytes.
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Affiliation(s)
- Cathleen E Berg
- Metabolic Diseases Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA
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45
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Zhande R, Mitchell JJ, Wu J, Sun XJ. Molecular mechanism of insulin-induced degradation of insulin receptor substrate 1. Mol Cell Biol 2002; 22:1016-26. [PMID: 11809794 PMCID: PMC134643 DOI: 10.1128/mcb.22.4.1016-1026.2002] [Citation(s) in RCA: 165] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Insulin receptor substrate 1 (IRS-1) plays an important role in the insulin signaling cascade. In vitro and in vivo studies from many investigators have suggested that lowering of IRS-1 cellular levels may be a mechanism of disordered insulin action (so-called insulin resistance). We previously reported that the protein levels of IRS-1 were selectively regulated by a proteasome degradation pathway in CHO/IR/IRS-1 cells and 3T3-L1 adipocytes during prolonged insulin exposure, whereas IRS-2 was unaffected. We have now studied the signaling events that are involved in activation of the IRS-1 proteasome degradation pathway. Additionally, we have addressed structural elements in IRS-1 versus IRS-2 that are required for its specific proteasome degradation. Using ts20 cells, which express a temperature-sensitive mutant of ubiquitin-activating enzyme E1, ubiquitination of IRS-1 was shown to be a prerequisite for insulin-induced IRS-1 proteasome degradation. Using IRS-1/IRS-2 chimeric proteins, the N-terminal region of IRS-1 including the PH and PTB domains was identified as essential for targeting IRS-1 to the ubiquitin-proteasome degradation pathway. Activation of phosphatidylinositol 3-kinase is necessary but not sufficient for activating and sustaining the IRS-1 ubiquitin-proteasome degradation pathway. In contrast, activation of mTOR is not required for IRS-1 degradation in CHO/IR cells. Thus, our data provide insight into the molecular mechanism of insulin-induced activation of the IRS-1 ubiquitin-proteasome degradation pathway.
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Affiliation(s)
- Rachel Zhande
- Endocrinology Division, University of Vermont College of Medicine, Burlington, Vermont 05405, USA
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46
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Richards RG, Klotz DM, Bush MR, Walmer DK, DiAugustine RP. E2-induced degradation of uterine insulin receptor substrate-2: requirement for an IGF-I-stimulated, proteasome-dependent pathway. Endocrinology 2001; 142:3842-9. [PMID: 11517161 DOI: 10.1210/endo.142.9.8370] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The insulin receptor substrates are docking proteins that bind various receptor tyrosine kinases and signaling proteins. Previous studies have shown that E2 or progesterone can regulate the relative abundance of insulin receptor substrate-1 and -2 in cells and tissues. For instance, uterine insulin receptor substrate-2 was decreased markedly at 24 h after E2 treatment of mice. In the present study we used various in vivo experimental approaches to examine the mechanism by which E2 influences uterine insulin receptor substrate-2 expression. Uterine insulin receptor substrate-2 mRNA levels were diminished after E2 treatment, but this diminution did not account for the total reduction in insulin receptor substrate-2 protein, suggesting that the E2-induced decrease in insulin receptor substrate-2 is not regulated solely at the mRNA level. Cotreatment with progesterone prevented the E2-stimulated reduction in insulin receptor substrate-2 protein at 24 h after hormone exposure. In addition, MG-132 and epoxomicin, inhibitors of proteasomal protease activity, inhibited the E2-induced decrease in uterine insulin receptor substrate-2 protein levels, and this correlated to an increase in uterine protein ubiquitination. Insulin receptor substrate-2 protein was diminished in uteri of E2-treated insulin receptor substrate-1-null mutant mice, but not in E2-treated IGF-I-null mutant mice. Furthermore, E2-induced diminution of uterine insulin receptor substrate-2 protein was only partially inhibited in the presence of wortmannin, a PI3K inhibitor. Collectively, these data suggest that the E2-induced decrease in uterine insulin receptor substrate-2 requires IGF-I signaling, is not dependent solely on insulin receptor substrate-1 and PI3K, and is blocked by progesterone as well as by pharmacological inhibition of proteasomal protease activity. We speculate that the IGF-I-activated IGF-I receptor, in response to E2, directly or indirectly modifies insulin receptor substrate-2, probably through phosphorylation, leading to ubiquitination and subsequent degradation of this docking protein by the proteasome. This degradation could be a regulatory step to inhibit insulin receptor substrate-2-dependent signaling in the uterus.
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Affiliation(s)
- R G Richards
- Hormones and Cancer Group, Laboratory of Molecular Carcinogenesis, National Institute of Environmental and Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA
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47
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Yu M, Blomstrand E, Chibalin AV, Wallberg-Henriksson H, Zierath JR, Krook A. Exercise-associated differences in an array of proteins involved in signal transduction and glucose transport. J Appl Physiol (1985) 2001; 90:29-34. [PMID: 11133890 DOI: 10.1152/jappl.2001.90.1.29] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Vastus lateralis muscle biopsies were obtained from endurance-trained (running approximately 50 km/wk) and untrained (no regular physical exercise) men, and the expression of an array of insulin-signaling intermediates was determined. Expression of insulin receptor and insulin receptor substrate-1 and -2 was decreased 44% (P < 0.05), 57% (P < 0.001), and 77% (P < 0.001), respectively, in trained vs. untrained muscle. The downstream signaling target, Akt kinase, was not altered in trained subjects. Components of the mitogenic signaling cascade were also assessed. Extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase expression was 190% greater (P < 0.05), whereas p38 mitogen-activated protein kinase expression was 32% lower (P < 0.05), in trained vs. untrained muscle. GLUT-4 protein expression was twofold higher (P < 0.05), and the GLUT-4 vesicle-associated protein, the insulin-regulated aminopeptidase, was increased 4.7-fold (P < 0. 05) in trained muscle. In conclusion, the expression of proteins involved in signal transduction is altered in skeletal muscle from well-trained athletes. Downregulation of early components of the insulin-signaling cascade may occur in response to increased insulin sensitivity associated with endurance training.
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Affiliation(s)
- M Yu
- Department of Clinical Physiology, Karolinska Hospital, SE-171 76 Stockholm, Sweden
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48
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Pederson TM, Kramer DL, Rondinone CM. Serine/threonine phosphorylation of IRS-1 triggers its degradation: possible regulation by tyrosine phosphorylation. Diabetes 2001; 50:24-31. [PMID: 11147790 DOI: 10.2337/diabetes.50.1.24] [Citation(s) in RCA: 232] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Insulin receptor substrate (IRS)-1 protein expression is markedly reduced in many insulin-resistant states, although the mechanism for this downregulation is unclear. In this study, we have investigated the early events in the insulin pathway that trigger the degradation of IRS-1. Incubation of the adipocytes with insulin induced a fast electrophoretic mobility shift of IRS-1 and a subsequent degradation of the protein. Wortmannin and rapamycin blocked this mobility shift of IRS-1, maintained the insulin-induced tyrosine phosphorylation of IRS-1, and blocked its degradation. In contrast, a glycogen synthase kinase 3 inhibitor, a mitogen-activated protein kinase/extracellular-regulated kinase inhibitor, and various protein kinase C inhibitors had no effect. Incubation with okadaic acid increased the serine/threonine phosphorylation of IRS-1 and its degradation, mimicking insulin, and its effect was prevented by the proteasome inhibitor lactacystin, as well as by rapamycin. Treatment of the cells with the tyrosine phosphatase inhibitor orthovanadate in the presence of insulin or okadaic acid partially inhibited the degradation of IRS-1. We propose that a rapamycin-dependent pathway participates as a negative regulator of IRS-1, increasing its serine/threonine phosphorylation, which triggers degradation. Thus, regulation of serine/threonine versus tyrosine phosphorylation may modulate IRS-1 degradation, affecting insulin sensitivity.
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Affiliation(s)
- T M Pederson
- Diabetes Research, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064-3500, USA
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49
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Zhang H, Hoff H, Sell C. Insulin-like growth factor I-mediated degradation of insulin receptor substrate-1 is inhibited by epidermal growth factor in prostate epithelial cells. J Biol Chem 2000; 275:22558-62. [PMID: 10811632 DOI: 10.1074/jbc.m000412200] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
We have sought to determine whether insulin-like growth factor I (IGF-I) regulates the levels of insulin receptor substrate-1 (IRS-1) in prostate epithelial cells. Exposure of prostate epithelial cells to IGF-I in the absence of other growth factors leads to a reduction in IRS-1 levels. Ubiquitin content of IRS-1 is increased in the presence of IGF-I, and inhibitors of the proteasome prevented the reduction of IRS-1 levels seen following IGF-I exposure. These results imply that IRS-1 is targeted to the proteasome upon exposure to IGF-I. The addition of epidermal growth factor (EGF) maintained IRS-1 levels even in the presence of IGF-I and inhibits IGF-I-dependent ubiquitination of IRS-1. Thus, these two growth factors, IGF-I and EGF, had antagonistic effects on IRS-1 protein levels in prostate epithelial cells. This regulation of IRS-1 reveals a novel level of cross-talk between the IGF-I and EGF signal pathways, which may have implications in tumors that harbor activating mutations in the EGF receptor.
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Affiliation(s)
- H Zhang
- Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096, USA
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50
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Haruta T, Uno T, Kawahara J, Takano A, Egawa K, Sharma PM, Olefsky JM, Kobayashi M. A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1. Mol Endocrinol 2000; 14:783-94. [PMID: 10847581 DOI: 10.1210/mend.14.6.0446] [Citation(s) in RCA: 318] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.
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Affiliation(s)
- T Haruta
- First Department of Medicine, Toyama Medical and Pharmaceutical University Japan.
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