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1
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Ahmad MM, Hassan HA, Saadawy SF, Ahmad EA, Elsawy NAM, Morsy MM. Antox targeting AGE/RAGE cascades to restore submandibular gland viability in rat model of type 1 diabetes. Sci Rep 2024; 14:18160. [PMID: 39103403 PMCID: PMC11300852 DOI: 10.1038/s41598-024-68268-z] [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: 10/08/2023] [Accepted: 07/22/2024] [Indexed: 08/07/2024] Open
Abstract
Diabetes mellitus (DM) is a chronic disorder of glucose metabolism that threatens several organs, including the submandibular (SMG) salivary glands. Antox (ANX) is a strong multivitamin with significant antioxidant benefits. The goal of this study was to demonstrate the beneficial roles of ANX supplementation in combination with insulin in alleviating diabetic SMG changes. For four weeks, 30 rats were divided into equal five groups (n = 6): (1) control group; (2) diabetic group (DM), with DM induced by streptozotocin (STZ) injection (50 mg/kg i.p.); (3) DM + ANX group: ANX was administrated (10 mg/kg/day/once daily/orally); (4) DM + insulin group: insulin was administrated 1U once/day/s.c.; and (5) DM + insulin + ANX group: co-administrated insulin. The addition of ANX to insulin in diabetic rats alleviated hyposalivation and histopathological alterations associated with diabetic rats. Remarkably, combined ANX and insulin exerted significant antioxidant effects, suppressing inflammatory and apoptotic pathways associated with increased salivary advanced glycation end-product (AGE) production and receptor for advanced glycation end-product expression (RAGE) activation in diabetic SMG tissues. Combined ANX and insulin administration in diabetic rats was more effective in alleviating SMG changes (functions and structures) than administration of insulin alone, exerting suppressive effects on AGE production and frustrating RAGE downstream pathways.
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Affiliation(s)
- Marwa M Ahmad
- Department of Human Anatomy and Embryology, Faculty of Medicine, Zagazig University, Zagazig, Egypt.
| | - Heba A Hassan
- Clinical Pharmacology Department, Faculty of Medicine, Zagazig University, Zagazig, 45519, Egypt
- Department of Pharmacology, Faculty of Medicine, Mutah University, Al-Karak, 61710, Jordan
| | - Sara F Saadawy
- Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt
| | - Enssaf Ahmad Ahmad
- Department of Human Anatomy and Embryology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
| | | | - Manal Mohammad Morsy
- Department of Human Anatomy and Embryology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
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2
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Jia JD, Jiang WG, Luo X, Li RR, Zhao YC, Tian G, Li YN. Vascular endothelial growth factor B inhibits insulin secretion in MIN6 cells and reduces Ca 2+ and cyclic adenosine monophosphate levels through PI3K/AKT pathway. World J Diabetes 2021; 12:480-498. [PMID: 33889292 PMCID: PMC8040075 DOI: 10.4239/wjd.v12.i4.480] [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: 12/08/2020] [Revised: 01/25/2021] [Accepted: 03/08/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Type 2 diabetes (T2D) is characterized by insufficient insulin secretion caused by defective pancreatic β-cell function or insulin resistance, resulting in an increase in blood glucose. However, the mechanism involved in this lack of insulin secretion is unclear. The level of vascular endothelial growth factor B (VEGF-B) is significantly increased in T2D patients. The inactivation of VEGF-B could restore insulin sensitivity in db/db mice by reducing fatty acid accumulation. It is speculated that VEGF-B is related to pancreatic β-cell dysfunction and is an important factor affecting β-cell secretion of insulin. As an in vitro model of normal pancreatic β-cells, the MIN6 cell line can be used to analyze the mechanism of insulin secretion and related biological effects.
AIM To study the role of VEGF-B in the insulin secretion signaling pathway in MIN6 cells and explore the effect of VEGF-B on blood glucose regulation.
METHODS The MIN6 mouse pancreatic islet β-cell line was used as the model system. By administering exogenous VEGF-B protein or knocking down VEGF-B expression in MIN6 cells, we examined the effects of VEGF-B on insulin secretion, Ca2+ and cyclic adenosine monophosphate (cAMP) levels, and the insulin secretion signaling pathway.
RESULTS Exogenous VEGF-B inhibited the secretion of insulin and simultaneously reduced the levels of Ca2+ and cAMP in MIN6 cells. Exogenous VEGF-B also reduced the expression of phospholipase C gamma 1 (PLCγ1), phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase (AKT), and other proteins in the insulin secretion pathway. Upon knockdown of VEGF-B, MIN6 cells exhibited increased insulin secretion and Ca2+ and cAMP levels and upregulated expression of PLCγ1, PI3K, AKT, and other proteins.
CONCLUSION VEGF-B can regulate insulin secretion by modulating the levels of Ca2+ and cAMP. VEGF-B involvement in insulin secretion is related to the expression of PLCγ1, PI3K, AKT, and other signaling proteins. These results provide theoretical support and an experimental basis for the study of VEGF-B in the pathogenesis of T2D.
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Affiliation(s)
- Jing-Dan Jia
- Department of Pathophysiology, School of Basic Medicine, Binzhou Medical University, Yantai 264003, Shandong Province, China
| | - Wen-Guo Jiang
- Department of Pharmacy, Binzhou Medical University, Yantai 264003, Shandong Province, China
| | - Xu Luo
- Department of Pathophysiology, School of Basic Medicine, Binzhou Medical University, Yantai 264003, Shandong Province, China
| | - Rong-Rong Li
- Department of Pathophysiology, School of Basic Medicine, Binzhou Medical University, Yantai 264003, Shandong Province, China
| | - Yu-Chi Zhao
- Department of Surgery, Yantaishan Hospital, Yantai 264001, Shandong Province, China
| | - Geng Tian
- Department of Pharmacy, Binzhou Medical University, Yantai 264003, Shandong Province, China
| | - Ya-Na Li
- Department of Pathophysiology, School of Basic Medicine, Binzhou Medical University, Yantai 264003, Shandong Province, China
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Han YE, Chun JN, Kwon MJ, Ji YS, Jeong MH, Kim HH, Park SH, Rah JC, Kang JS, Lee SH, Ho WK. Endocytosis of K ATP Channels Drives Glucose-Stimulated Excitation of Pancreatic β Cells. Cell Rep 2019; 22:471-481. [PMID: 29320741 DOI: 10.1016/j.celrep.2017.12.049] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2017] [Revised: 11/02/2017] [Accepted: 12/14/2017] [Indexed: 11/30/2022] Open
Abstract
Insulin secretion from pancreatic β cells in response to high glucose (HG) critically depends on the inhibition of KATP channel activity in HG. It is generally believed that HG-induced effects are mediated by the increase in intracellular ATP, but here, we showed that, in INS-1 cells, endocytosis of KATP channel plays a major role. Upon HG stimulation, resting membrane potential depolarized by 30.6 mV (from -69.2 to -38.6 mV) and KATP conductance decreased by 91% (from 0.243 to 0.022 nS/pF), whereas intracellular ATP was increased by only 47%. HG stimulation induced internalization of KATP channels, causing a significant decrease in surface channel density, and this decrease was completely abolished by inhibiting endocytosis using dynasore, a dynamin inhibitor, or a PKC inhibitor. These drugs profoundly inhibited HG-induced depolarization. Our results suggest that the control of KATP channel surface density plays a greater role than ATP-dependent gating in regulating β cell excitability.
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Affiliation(s)
- Young-Eun Han
- Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Biomembrane Plasticity Research Center, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Korea Brain Research Institute, Daegu 41068, Republic of Korea
| | - Jung Nyeo Chun
- Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
| | - Min Jeong Kwon
- Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Biomembrane Plasticity Research Center, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
| | - Young-Sun Ji
- Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Biomembrane Plasticity Research Center, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
| | - Myong-Ho Jeong
- Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Republic of Korea
| | - Hye-Hyun Kim
- Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Biomembrane Plasticity Research Center, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
| | - Sun-Hyun Park
- Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Biomembrane Plasticity Research Center, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
| | - Jong Cheol Rah
- Korea Brain Research Institute, Daegu 41068, Republic of Korea
| | - Jong-Sun Kang
- Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Republic of Korea
| | - Suk-Ho Lee
- Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Biomembrane Plasticity Research Center, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
| | - Won-Kyung Ho
- Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea; Biomembrane Plasticity Research Center, Seoul National University College of Medicine, Seoul 03080, Republic of Korea.
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4
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Aljaibeji H, Mukhopadhyay D, Mohammed AK, Dhaiban S, Hachim MY, Elemam NM, Sulaiman N, Salehi A, Taneera J. Reduced Expression of PLCXD3 Associates With Disruption of Glucose Sensing and Insulin Signaling in Pancreatic β-Cells. Front Endocrinol (Lausanne) 2019; 10:735. [PMID: 31781030 PMCID: PMC6851018 DOI: 10.3389/fendo.2019.00735] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/12/2019] [Accepted: 10/10/2019] [Indexed: 01/17/2023] Open
Abstract
Previous work has shown that reduced expression of PLCXD3, a member of the phosphoinositide-specific phospholipases (PI-PLC) family, impaired insulin secretion with an unclear mechanism. In the current study, we aim to investigate the mechanism underlying this effect using human islets and rat INS-1 (832/13) cells. Microarray and RNA sequencing data showed that PLCXD3 is among the highly expressed PI-PLCs in human islets and INS-1 (832/13) cells. Expression of PLCXD3 was reduced in human diabetic islets, correlated positively with Insulin and GLP1R expression and inversely with the donor's body mass index (BMI) and glycated hemoglobin (HbA1c). Expression silencing of PLCXD3 in INS-1 (832/13) cells was found to reduce glucose-stimulated insulin secretion (GSIS) and insulin content. In addition, the expression of Insulin, NEUROD1, GLUT2, GCK, INSR, IRS2, and AKT was downregulated. Cell viability and apoptosis rate were unaffected. In conclusion, our data suggest that low expression of PLCXD3 in pancreatic β-cells associates with downregulation of the key insulin signaling and insulin biosynthesis genes as well as reduction in glucose sensing.
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Affiliation(s)
- Hayat Aljaibeji
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
| | - Debasmita Mukhopadhyay
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
| | - Abdul Khader Mohammed
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
| | - Sarah Dhaiban
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
| | - Mahmood Y. Hachim
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
| | - Noha M. Elemam
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
| | - Nabil Sulaiman
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
| | - Albert Salehi
- Department of Clinical Sciences, Lund University Diabetes Centre (LUDC), Lund University, Malmö, Sweden
| | - Jalal Taneera
- Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates
- *Correspondence: Jalal Taneera
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5
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Trexler AJ, Taraska JW. Regulation of insulin exocytosis by calcium-dependent protein kinase C in beta cells. Cell Calcium 2017; 67:1-10. [PMID: 29029784 DOI: 10.1016/j.ceca.2017.07.008] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2017] [Revised: 07/25/2017] [Accepted: 07/27/2017] [Indexed: 12/27/2022]
Abstract
The control of insulin release from pancreatic beta cells helps ensure proper blood glucose level, which is critical for human health. Protein kinase C has been shown to be one key control mechanism for this process. After glucose stimulation, calcium influx into beta cells triggers exocytosis of insulin-containing dense-core granules and activates protein kinase C via calcium-dependent phospholipase C-mediated generation of diacylglycerol. Activated protein kinase C potentiates insulin release by enhancing the calcium sensitivity of exocytosis, likely by affecting two main pathways that could be linked: (1) the reorganization of the cortical actin network, and (2) the direct phosphorylation of critical exocytotic proteins such as munc18, SNAP25, and synaptotagmin. Here, we review what is currently known about the molecular mechanisms of protein kinase C action on each of these pathways and how these effects relate to the control of insulin release by exocytosis. We identify remaining challenges in the field and suggest how these challenges might be addressed to advance our understanding of the regulation of insulin release in health and disease.
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Affiliation(s)
- Adam J Trexler
- National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, United States
| | - Justin W Taraska
- National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, United States.
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6
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Seed Ahmed M, Ahmed MS, Pelletier J, Leumann H, Gu HF, Östenson CG. Expression of Protein Kinase C Isoforms in Pancreatic Islets and Liver of Male Goto-Kakizaki Rats, a Model of Type 2 Diabetes. PLoS One 2015; 10:e0135781. [PMID: 26398746 PMCID: PMC4580567 DOI: 10.1371/journal.pone.0135781] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2014] [Accepted: 07/27/2015] [Indexed: 02/03/2023] Open
Abstract
Protein kinase C (PKC) is a family of protein kinases controlling protein phosphorylation and playing important roles in the regulation of metabolism. We have investigated expression levels of PKC isoforms in pancreatic islets and liver of diabetic Goto-Kakizaki (GK) rats with and without insulin treatment to evaluate their association with glucose homeostasis. mRNA and protein expression levels of PKC isoforms were assessed in pancreatic islets and liver of Wistar rats and GK rats with or without insulin treatment. PKCα and PKCζ mRNA expressions were down-regulated in islets of GK compared with Wistar rats. PKCα and phosphorylated PKCα (p-PKCα) protein expressions were decreased in islets of GK compared with insulin-treated GK and Wistar rats. PKCζ protein expression in islets was reduced in GK and insulin-treated GK compared with Wistar rats, but p-PKCζ was decreased only in GK rats. Islet PKCε mRNA and protein expressions were lower in GK compared with insulin-treated GK and Wistar rats. In liver, PKCδ and PKCζ mRNA expressions were decreased in both GK and insulin-treated GK compared with Wistar rats. Hepatic PKCζ protein expression was diminished in both GK rats with and without insulin treatment compared with Wistar rats. Hepatic PKCε mRNA expression was down-regulated in insulin-treated GK compared with GK and Wistar rats. PKCα, PKCε, and p-PKCζ expressions were secondary to hyperglycaemia in GK rat islets. Hepatic PKCδ and PKCζ mRNA expressions were primarily linked to hyperglycaemia. Additionally, hepatic PKCε mRNA expression could be under control of insulin.
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Affiliation(s)
- Mohammed Seed Ahmed
- Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden; Department of Physiology, Faculty of Medicine, University of Khartoum, Khartoum, Sudan
| | | | - Julien Pelletier
- Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden
| | - Hannes Leumann
- Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden
| | - Harvest F Gu
- Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden
| | - Claes-Göran Östenson
- Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden
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7
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Taneera J, Fadista J, Ahlqvist E, Atac D, Ottosson-Laakso E, Wollheim CB, Groop L. Identification of novel genes for glucose metabolism based upon expression pattern in human islets and effect on insulin secretion and glycemia. Hum Mol Genet 2014; 24:1945-55. [PMID: 25489054 DOI: 10.1093/hmg/ddu610] [Citation(s) in RCA: 82] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Normal glucose homeostasis is characterized by appropriate insulin secretion and low HbA1c. Gene expression signatures associated with these two phenotypes could be essential for islet function and pathophysiology of type 2 diabetes (T2D). Herein, we employed a novel approach to identify candidate genes involved in T2D by correlating islet microarray gene expression data (78 donors) with insulin secretion and HbA1c level. The expression of 649 genes (P < 0.05) was correlated with insulin secretion and HbA1c. Of them, five genes (GLR1A, PPP1R1A, PLCDXD3, FAM105A and ENO2) correlated positively with insulin secretion/negatively with HbA1c and one gene (GNG5) correlated negatively with insulin secretion/positively with HbA1c were followed up. The five positively correlated genes have lower expression levels in diabetic islets, whereas GNG5 expression is higher. Exposure of human islets to high glucose for 24 h resulted in up-regulation of GNG5 and PPP1R1A expression, whereas the expression of ENO2 and GLRA1 was down-regulated. No effect was seen on the expression of FAM105A and PLCXD3. siRNA silencing in INS-1 832/13 cells showed reduction in insulin secretion for PPP1R1A, PLXCD3, ENO2, FAM105A and GNG5 but not GLRA1. Although no SNP in these gene loci passed the genome-wide significance for association with T2D in DIAGRAM+ database, four SNPs influenced gene expression in cis in human islets. In conclusion, we identified and confirmed PPP1R1A, FAM105A, ENO2, PLCDX3 and GNG5 as potential regulators of islet function. We provide a list of candidate genes as a resource for exploring their role in the pathogenesis of T2D.
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Affiliation(s)
- Jalal Taneera
- Department of Clinical Sciences, Diabetes and Endocrinology, Lund University Diabetes Centre, Skåne University Hospital, Lund University, 205 02 Malmö, Sweden
| | - Joao Fadista
- Department of Clinical Sciences, Diabetes and Endocrinology, Lund University Diabetes Centre, Skåne University Hospital, Lund University, 205 02 Malmö, Sweden
| | - Emma Ahlqvist
- Department of Clinical Sciences, Diabetes and Endocrinology, Lund University Diabetes Centre, Skåne University Hospital, Lund University, 205 02 Malmö, Sweden
| | - David Atac
- Department of Clinical Sciences, Diabetes and Endocrinology, Lund University Diabetes Centre, Skåne University Hospital, Lund University, 205 02 Malmö, Sweden
| | - Emilia Ottosson-Laakso
- Department of Clinical Sciences, Diabetes and Endocrinology, Lund University Diabetes Centre, Skåne University Hospital, Lund University, 205 02 Malmö, Sweden
| | - Claes B Wollheim
- Department of Clinical Sciences, Diabetes and Endocrinology, Lund University Diabetes Centre, Skåne University Hospital, Lund University, 205 02 Malmö, Sweden Department of Cell Physiology and Metabolism, Université de Genève, University Medical Centre, 1 rue Michel-Servet, Geneva 4 1211, Switzerland
| | - Leif Groop
- Department of Clinical Sciences, Diabetes and Endocrinology, Lund University Diabetes Centre, Skåne University Hospital, Lund University, 205 02 Malmö, Sweden
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Jonatan D, Spence JR, Method AM, Kofron M, Sinagoga K, Haataja L, Arvan P, Deutsch GH, Wells JM. Sox17 regulates insulin secretion in the normal and pathologic mouse β cell. PLoS One 2014; 9:e104675. [PMID: 25144761 PMCID: PMC4140688 DOI: 10.1371/journal.pone.0104675] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2014] [Accepted: 07/16/2014] [Indexed: 02/06/2023] Open
Abstract
SOX17 is a key transcriptional regulator that can act by regulating other transcription factors including HNF1β and FOXA2, which are known to regulate postnatal β cell function. Given this, we investigated the role of SOX17 in the developing and postnatal pancreas and found a novel role for SOX17 in regulating insulin secretion. Deletion of the Sox17 gene in the pancreas (Sox17-paLOF) had no observable impact on pancreas development. However, Sox17-paLOF mice had higher islet proinsulin protein content, abnormal trafficking of proinsulin, and dilated secretory organelles suggesting that Sox17-paLOF adult mice are prediabetic. Consistant with this, Sox17-paLOF mice were more susceptible to aged-related and high fat diet-induced hyperglycemia and diabetes. Overexpression of Sox17 in mature β cells using Ins2-rtTA driver mice resulted in precocious secretion of proinsulin. Transcriptionally, SOX17 appears to broadly regulate secretory networks since a 24-hour pulse of SOX17 expression resulted in global transcriptional changes in factors that regulate hormone transport and secretion. Lastly, transient SOX17 overexpression was able to reverse the insulin secretory defects observed in MODY4 animals and restored euglycemia. Together, these data demonstrate a critical new role for SOX17 in regulating insulin trafficking and secretion and that modulation of Sox17-regulated pathways might be used therapeutically to improve cell function in the context of diabetes.
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Affiliation(s)
- Diva Jonatan
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States of America
| | - Jason R. Spence
- Department of Internal Medicine, Division of Gastroenterology, University of Michigan Medical School, Ann Arbor, MI, United States of America
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States of America
- Center for Organogenesis, University of Michigan Medical School, Ann Arbor, MI, United States of America
| | - Anna M. Method
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States of America
| | - Matthew Kofron
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States of America
| | - Katie Sinagoga
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States of America
| | - Leena Haataja
- Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, MI, United States of America
| | - Peter Arvan
- Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, MI, United States of America
| | - Gail H. Deutsch
- Seattle Children’s Hospital, Seattle, WA, United States of America
| | - James M. Wells
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States of America
- Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States of America
- * E-mail:
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9
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Inoue T, Hagiyama M, Yoneshige A, Kato T, Enoki E, Maenishi O, Chikugo T, Kimura M, Satou T, Ito A. Increased ectodomain shedding of cell adhesion molecule 1 from pancreatic islets in type 2 diabetic pancreata: correlation with hemoglobin A1c levels. PLoS One 2014; 9:e100988. [PMID: 24964098 PMCID: PMC4071031 DOI: 10.1371/journal.pone.0100988] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2014] [Accepted: 05/31/2014] [Indexed: 01/09/2023] Open
Abstract
Pulmonary emphysema and type 2 diabetes mellitus (T2DM), both caused by lifestyle factors, frequently concur. Respectively, the diseases affect lung alveolar and pancreatic islet cells, which express cell adhesion molecule 1 (CADM1), an immunoglobulin superfamily member. Protease-mediated ectodomain shedding of full-length CADM1 produces C-terminal fragments (CTFs) with proapoptotic activity. In emphysematous lungs, the CADM1 shedding rate and thus the level of CTFs in alveolar cells increase. In this study, CADM1 expression in islet cells was examined by western blotting. Protein was extracted from formalin-fixed, paraffin-embedded sections of pancreata isolated from patients with T2DM (n = 12) or from patients without pancreatic disease (n = 8) at autopsy. After adjusting for the number of islet cells present in the adjacent section, we found that full-length CADM1 decreased in T2DM islets, while ectodomain shedding increased. Hemoglobin A1c levels, measured when patients were alive, correlated inversely with full-length CADM1 levels (P = 0.041) and positively with ectodomain shedding rates (P = 0.001). In immunofluorescence images of T2DM islet cells, CADM1 was detected in the cytoplasm, but not on the cell membrane. Consistently, when MIN6-m9 mouse beta cells were treated with phorbol ester and trypsin to induce shedding, CADM1 immunostaining was diffuse in the cytoplasm. When a form of CTFs was exogenously expressed in MIN6-m9 cells, it localized diffusely in the cytoplasm and increased the number of apoptotic cells. These results suggest that increased CADM1 ectodomain shedding contributes to blood glucose dysregulation in T2DM by decreasing full-length CADM1 and producing CTFs that accumulate in the cytoplasm and promote apoptosis of beta cells. Thus, this study has identified a molecular alteration shared by pulmonary emphysema and T2DM.
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Affiliation(s)
- Takao Inoue
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
| | - Man Hagiyama
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
| | - Azusa Yoneshige
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
| | - Takashi Kato
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
| | - Eisuke Enoki
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
| | - Osamu Maenishi
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
| | - Takaaki Chikugo
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
| | - Masatomo Kimura
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
| | - Takao Satou
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
| | - Akihiko Ito
- Department of Pathology, Faculty of Medicine, Kinki University, Osaka, Japan
- * E-mail:
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10
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Fiume R, Ramazzotti G, Faenza I, Piazzi M, Bavelloni A, Billi AM, Cocco L. Nuclear PLCs affect insulin secretion by targeting PPARγ in pancreatic β cells. FASEB J 2011; 26:203-10. [PMID: 21974932 DOI: 10.1096/fj.11-186510] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Type 2 diabetes is a heterogeneous disorder caused by concomitant impairment of insulin secretion by pancreatic β cells and of insulin action in peripheral target tissues. Studies with inhibitors and agonists established a role for PLC in the regulation of insulin secretion but did not distinguish between effects due to nuclear or cytoplasmic PLC signaling pathways that act in a distinct fashion. We report that in MIN6 β cells, PLCβ1 localized in both nucleus and cytoplasm, PLCδ4 in the nucleus, and PLCγ1 in the cytoplasm. By silencing each isoform, we observed that they all affected glucose-induced insulin release both at basal and high glucose concentrations. To elucidate the molecular basis of PLC regulation, we focused on peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor transcription factor that regulates genes critical to β-cell maintenance and functions. Silencing of PLCβ1 and PLCδ4 resulted in a decrease in the PPARγ mRNA level. By means of a PPARγ-promoter-luciferase assay, the decrease could be attributed to a PLC action on the PPARγ-promoter region. The effect was specifically observed on silencing of the nuclear and not the cytoplasmic PLC. These findings highlight a novel pathway by which nuclear PLCs affect insulin secretion and identify PPARγ as a novel molecular target of nuclear PLCs.
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Affiliation(s)
- Roberta Fiume
- Department of Human Anatomy, University of Bologna, Bologna, Italy
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11
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Kang N, Won JH, Park YM. Annexin I stimulates insulin secretion through regulation of cytoskeleton and PKC activity. Anim Cells Syst (Seoul) 2010. [DOI: 10.1080/19768354.2009.9647190] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
Affiliation(s)
- Na‐na Kang
- a Department of Biological Sciences and Institute for Basic Sciences , Sungkyunkwan University , Suwon, 440–746, Korea
| | - Jong Hak Won
- b Department of Pharmacology and Physiology , University of Rochester , Rochester, New York, 14642, USA
| | - Young Min Park
- c Department of Biological Sciences and Institute for Basic Sciences , Sungkyunkwan University , Suwon, 440–746, Korea Phone: Fax: E-mail:
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12
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Yamazaki H, Zawalich KC, Zawalich WS. Physiologic implications of phosphoinositides and phospholipase C in the regulation of insulin secretion. J Nutr Sci Vitaminol (Tokyo) 2010; 56:1-8. [PMID: 20354339 DOI: 10.3177/jnsv.56.1] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
The secretion of insulin from the pancreatic beta-cell must be commensurate to satisfy the insulin requirements of the organism. This cell has a great flexibility to meet these requirements which are increased not only by the ingestion of nutrients (increase of plasma glucose) but also by the sensitivity of target tissues to insulin as well. The insulin secretion is a complex biochemical event regulated by a host of potential second messenger molecules acting alone or in concert. These events include the cation calcium, which gains access to the beta-cell via the opening of voltage-regulated channels, cAMP and phosphoinositide-derived second messenger molecules, generated as a consequence of phospholipase C (PLC) activation. In this review, we focused on phosphoinositides, PLC/Phosphokinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) cascade in the regulation of insulin secretion. We also described our studies on the mechanism of the beta-cell desensitization using perifused islets. It is suggested that a failure of the signaling events contribute to the pathogenesis of diabetes in which the beta-cell can no longer secrete the required amounts of insulin. It has been observed that chronic exposure to high glucose desensitizes the beta-cells to subsequent stimulation. We suggested that the failure of PLC activation can be attributed in the impairment of insulin secretion by chronic sustained glucose exposure. It may contribute to the vicious circle of impaired insulin secretion leading up to diabetes.
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Affiliation(s)
- Hanae Yamazaki
- Laboratory of Ajinomoto Integrative Research for Advanced Dieting, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
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13
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Zawalich WS, Yamazaki H, Zawalich KC. Biphasic insulin secretion from freshly isolated or cultured, perifused rodent islets: comparative studies with rats and mice. Metabolism 2008; 57:30-9. [PMID: 18078856 PMCID: PMC2214880 DOI: 10.1016/j.metabol.2007.07.020] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/16/2007] [Accepted: 07/23/2007] [Indexed: 11/19/2022]
Abstract
In the present report, we compared the insulin secretory responses of freshly isolated, perifused rat and mouse islets to glucose. Prestimulatory glucose levels were changed to assess their influence on the subsequent secretory responses. Additional studies included experiments with the incretin factor glucagon-like peptide-1 (GLP-1), the cholinergic agonist carbachol, and the alpha2 agonist epinephrine. Our findings demonstrate that under conditions where glucose (8.5-11.1 mmol/L) evokes a dramatic biphasic insulin secretory response from perifused rat islets, mouse islets exhibit little response. Increasing the prestimulatory glucose level to 8.5 mmol/L dramatically distorts subsequently measured glucose-induced insulin secretion from rat islets but allows the evocation of a modest but clear biphasic response from mouse islets in response to 30 mmol/L, but not 11.1 or 16.7 mmol/L, glucose. In the presence of a minimally effective glucose level (10 mmol/L), mouse islets remain exquisitely sensitive to the combined stimulatory effects of GLP-1 (2.5 nmol/L) plus carbachol (0.5 micromol/L) and to the inhibitory influence of epinephrine (10 nmol/L). Short-term culture of rat islets in CMRL 1066 containing 5.6 mmol/L glucose resulted in a significant decrease in the secretory response to 11.1 mmol/L glucose, whereas the same manipulation improved mouse islet responses. It is concluded that the process of collagenase isolating islets does not alter mouse islet sensitivity in any adverse way and that increasing the prestimulatory glucose level can indeed alter the pattern of insulin secretion in either a positive or negative manner depending upon the species being investigated. Prior short-term culture of rodent islets differentially affects secretion from these 2 species.
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14
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Uchida T, Iwashita N, Ohara-Imaizumi M, Ogihara T, Nagai S, Choi JB, Tamura Y, Tada N, Kawamori R, Nakayama KI, Nagamatsu S, Watada H. Protein Kinase Cδ Plays a Non-redundant Role in Insulin Secretion in Pancreatic β Cells. J Biol Chem 2007; 282:2707-16. [PMID: 17135234 DOI: 10.1074/jbc.m610482200] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Protein kinase C (PKC) is considered to modulate glucose-stimulated insulin secretion. Pancreatic beta cells express multiple isoforms of PKCs; however, the role of each isoform in glucose-stimulated insulin secretion remains controversial. In this study we investigated the role of PKCdelta, a major isoform expressed in pancreatic beta cells on beta cell function. Here, we showed that PKCdelta null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKCdelta null mice. Studies using islets isolated from these mice demonstrated decreased glucose- and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca2+ into beta cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in beta cells of PKCdelta null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKCdelta null mice. We conclude that PKCdelta plays a non-redundant role in glucose-stimulated insulin secretion. The impaired insulin secretion in PKCdelta null mice is associated with reduced phosphorylation of Munc18-1.
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Affiliation(s)
- Toyoyoshi Uchida
- Department of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine, 2-1-1, Tokyo 113-8421, Japan
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15
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Warwar N, Efendic S, Ostenson CG, Haber EP, Cerasi E, Nesher R. Dynamics of glucose-induced localization of PKC isoenzymes in pancreatic beta-cells: diabetes-related changes in the GK rat. Diabetes 2006; 55:590-9. [PMID: 16505220 DOI: 10.2337/diabetes.55.03.06.db05-0001] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Glucose metabolism affects most major signal pathways in pancreatic beta-cells. Multiple protein kinases, including protein kinase C (PKC) isoenzymes, are involved in these effects; however, their role is poorly defined. Moreover, the dynamics of kinase isoenzyme activation in reference to the biphasic insulin secretion is unknown. In perfused pancreas of Wistar rats, PKCalpha staining was strongly associated with insulin staining, jointly accumulating in the vicinity of the plasma membrane during early first-phase insulin response. The signal declined before the onset of second phase and reappeared during second-phase insulin release as foci, only weekly associated with insulin staining; this signal persisted for at least 15 min after glucose stimulation. In the GK rat, glucose had minimal effect on beta-cell PKCalpha. In control beta-cells, PKCdelta stained as granulated foci with partial association with insulin staining; however, no glucose-dependent translocation was observed. In the GK rat, only minimal staining for PKCdelta was observed, increasing exclusively during early first-phase secretion. In Wistar beta-cells, PKCepsilon concentrated near the nucleus, strongly associated with insulin staining, with dynamics resembling that of biphasic insulin response, but persisting for 15 min after cessation of stimulation. In GK rats, PKCepsilon staining lacked glucose-dependent changes or association with insulin. PKCzeta exhibited bimodal dynamics in control beta-cells: during early first phase, accumulation near the cell membrane was observed, dispersing thereafter. This was followed by a gradual accumulation near the nucleus; 15 min after glucose stimulus, clear PKCzeta staining was observed within the nucleus. In the GK rat, a similar response was only occasionally observed. In control beta-cells, glucose stimulation led to a transient recruitment of PKCtheta, associated with first-phase insulin release, not seen in GK beta-cell. Data from this and related studies support a role for PKCalpha in glucose-induced insulin granule recruitment for exocytosis; a role for PKCepsilon in activation of insulin granules for exocytosis and/or in the glucose-generated time-dependent potentiation signal for insulin release; and a dual function for PKCzeta in initiating insulin release and in a regulatory role in the transcriptional machinery. Furthermore, diminished levels and/or activation of PKCalpha, PKCepsilon, PKCtheta, and PKCzeta could be part of the defective signals downstream to glucose metabolism responsible for the deranged insulin secretion in the GK rat.
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Affiliation(s)
- Nasim Warwar
- Endocrinology and Metabolism Service, Hebrew University, Hadassah Medical Center, Jerusalem, Israel
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16
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Zawalich WS, Tesz GJ, Yamazaki H, Zawalich KC, Philbrick W. Dexamethasone suppresses phospholipase C activation and insulin secretion from isolated rat islets. Metabolism 2006; 55:35-42. [PMID: 16324917 DOI: 10.1016/j.metabol.2005.06.023] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/30/2005] [Accepted: 06/24/2005] [Indexed: 11/21/2022]
Abstract
Dexamethasone inhibits insulin secretion from isolated islets. In the present experiments, possible underlying biochemical mechanisms responsible for defective secretion were explored. Dexamethasone (1 micromol/L) had no immediate deleterious effect on 15 mmol/L glucose-induced insulin release from perifused rat islets. However, a 3-hour preincubation period with 1 micromol/L dexamethasone resulted in parallel reductions in both the first (64%) and second phases (74%) of 15 mmol/L glucose-induced insulin secretion monitored during a dynamic perifusion. When measured after the perifusion, there were no differences in insulin content or in the capacity of control or dexamethasone-treated islets to use glucose. Dexamethasone (1 micromol/L) preexposure also reduced phorbol ester- and potassium-induced secretion. In additional experiments, islets were labeled for 3 hours with 3H-inositol in the presence or absence of 1 micromol/L dexamethasone. The steroid did not affect total 3H-inositol incorporation during the labeling period. However, the capacity of 15 mmol/L glucose, 30 mmol/L KCl, and 100 micromol/L carbachol to activate phospholipase C (PLC), monitored by the accumulation of labeled inositol phosphates, was significantly reduced in dexamethasone-pretreated islets. Inclusion of the nuclear glucocorticoid receptor antagonist RU486 (mifepristone, 10 micromol/L) abolished the adverse effects of dexamethasone on both glucose-induced inositol phosphate accumulation and insulin secretion. Quantitative Western blot analyses revealed that the islet contents of PLCdelta1, PLCbeta1, beta2, beta3, and protein kinase C alpha were unaffected by dexamethasone pretreatment. These findings demonstrate that dexamethasone pretreatment impairs insulin secretion via a genomic action and that impaired activation of the PLC/protein kinase C signaling system is involved in the evolution of its inhibitory effect on secretion.
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17
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Akiba Y, Kato S, Katsube KI, Nakamura M, Takeuchi K, Ishii H, Hibi T. Transient receptor potential vanilloid subfamily 1 expressed in pancreatic islet beta cells modulates insulin secretion in rats. Biochem Biophys Res Commun 2004; 321:219-25. [PMID: 15358238 DOI: 10.1016/j.bbrc.2004.06.149] [Citation(s) in RCA: 130] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2004] [Indexed: 10/26/2022]
Abstract
Capsaicin-sensitive afferent neurons including transient receptor potential vanilloid subfamily 1, TRPV1, and neurohormonal peptides participate in the physiological regulation of pancreatic endocrine. However, the direct effect of capsaicin on insulin secretion remains unknown. Our present study showed that TRPV1 is expressed in islet beta cells as well as in neurons in rat pancreas, and also in rat beta cell lines, RIN and INS1. Capsaicin (10(-11)-10(-9) M) dose-dependently increased insulin secretion from RIN cells, and this effect was inhibited by either a TRPV1 inhibitor capsazepine or EDTA. Systemic capsaicin (10 mg/kg, s.c.) increased plasma insulin level 1 h after the treatment. We demonstrated for the first time that TRPV1 is functionally expressed in rat islet beta cells and plays a role in insulin secretion as a calcium channel. This study may account for the influences of capsaicin on the food intake and energy consumption as well as on the pathophysiological regulation of pancreatic endocrine.
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Affiliation(s)
- Yasutada Akiba
- Department of Internal Medicine, School of Medicine, Keio University, Tokyo 160-8582, Japan.
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18
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Zhang H, Nagasawa M, Yamada S, Mogami H, Suzuki Y, Kojima I. Bimodal role of conventional protein kinase C in insulin secretion from rat pancreatic beta cells. J Physiol 2004; 561:133-47. [PMID: 15388777 PMCID: PMC1665327 DOI: 10.1113/jphysiol.2004.071241] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Abstract
The present study was conducted to evaluate the role of conventional protein kinase C (PKC) in calcium-evoked insulin secretion. In rat beta cells transfected with green fluorescent protein-tagged PKC-alpha (PKC-alpha-EGFP), a depolarizing concentration of potassium induced transient elevation of cytoplasmic free calcium ([Ca(2)(+)](c)), which was accompanied by transient translocation of PKC-alpha-EGFP from the cytosol to the plasma membrane. Potassium also induced transient translocation of PKC-theta-EGFP, the C1 domain of PKC-gamma and PKC-epsilon-GFP. A high concentration of glucose induced repetitive elevation of [Ca(2)(+)](c) and repetitive translocation of PKC-alpha-EGFP. Diazoxide completely blocked both elevation of [Ca(2)(+)](c) and translocation of PKC-alpha-EGFP. We then studied the role of conventional PKC in calcium-evoked insulin secretion using rat islets. When islets were incubated for 10 min with high potassium, Go-6976, an inhibitor of conventional PKC, and PKC-alpha pseudosubstrate fused to antennapedia peptide (Antp-PKC(19-31)) increased potassium induced secretion. Similarly, insulin release induced by high glucose for 10 min was enhanced by Gö-6976 and Antp-PKC(19-31). However, when islets were stimulated for 60 min with high glucose, both Gö-6976 and Antp-PKC(19-31) reduced glucose-induced insulin secretion. Similar results were obtained by transfection of dominant-negative PKC-alpha using adenovirus vector. Taken together, PKC-alpha is activated when cells are depolarized by a high concentration of potassium or glucose. Conventional PKC is inhibitory on depolarization-induced insulin secretion per se, but it also augments glucose-induced secretion.
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Affiliation(s)
- Hui Zhang
- Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
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19
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Carpenter L, Mitchell CJ, Xu ZZ, Poronnik P, Both GW, Biden TJ. PKC alpha is activated but not required during glucose-induced insulin secretion from rat pancreatic islets. Diabetes 2004; 53:53-60. [PMID: 14693697 DOI: 10.2337/diabetes.53.1.53] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The role of protein kinase C (PKC) in glucose-stimulated insulin secretion (GSIS) is controversial. Using recombinant adenoviruses for overexpression of PKC alpha and PKC delta, in both wild-type (WT) and kinase-dead (KD) forms, we here demonstrate that activation of these two PKCs is neither necessary nor sufficient for GSIS from batch-incubated, rat pancreatic islets. In contrast, responses to the pharmacologic activator 12-O-tetradecanoylphorbol-13-acetate (TPA) were reciprocally modulated by overexpression of the PKC alpha WT or PKC alpha KD but not the corresponding PKC delta adenoviruses. The kinetics of the secretory response to glucose (monitored by perifusion) were not altered in either cultured islets overexpressing PKC alpha KD or freshly isolated islets stimulated in the presence of the conventional PKC (cPKC) inhibitor Go6976. However, the latter did inhibit the secretory response to TPA. Using phosphorylation state-specific antisera for consensus PKC phosphorylation sites, we also showed that (compared with TPA) glucose causes only a modest and transient functional activation of PKC (maximal at 2-5 min). However, glucose did promote a prolonged (15 min) phosphorylation of PKC substrates in the presence of the phosphatase inhibitor okadaic acid. Overall, the results demonstrate that glucose does stimulate PKC alpha in pancreatic islets but that this makes little overall contribution to GSIS.
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Affiliation(s)
- Lee Carpenter
- Garvan Institute of Medical Research, St. Vincents Hospital, and Department of Medicine, University of New South Wales, Sydney, Australia
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20
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Mendez CF, Leibiger IB, Leibiger B, Høy M, Gromada J, Berggren PO, Bertorello AM. Rapid association of protein kinase C-epsilon with insulin granules is essential for insulin exocytosis. J Biol Chem 2003; 278:44753-7. [PMID: 12941947 DOI: 10.1074/jbc.m308664200] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Glucose-dependent exocytosis of insulin requires activation of protein kinase C (PKC). However, because of the great variety of isoforms and their ubiquitous distribution within the beta-cell, it is difficult to predict the importance of a particular isoform and its mode of action. Previous data revealed that two PKC isoforms (alpha and epsilon) translocate to membranes in response to glucose (Zaitzev, S. V., Efendic, S., Arkhammar, P., Bertorello, A. M., and Berggren, P. O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9712-9716). Using confocal microscopy, we have now established that in response to glucose, PKC-epsilon but not PKC-alpha associates with insulin granules and that green fluorescent protein-tagged PKC-epsilon changes its distribution within the cell periphery upon stimulation of beta-cells with glucose. Definite evidence of PKC-epsilon requirement during insulin granule exocytosis was obtained by using a dominant negative mutant of this isoform. The presence of this mutant abolished glucose-induced insulin secretion, whereas transient expression of the wild-type PKC-epsilon led to a significant increase in insulin exocytosis. These results suggest that association of PKC-epsilon with insulin granule membranes represents an important component of the secretory network because it is essential for insulin exocytosis in response to glucose.
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Affiliation(s)
- Carlos F Mendez
- Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, SE-171 76 Stockholm, Sweden
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21
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Yaney GC, Corkey BE. Fatty acid metabolism and insulin secretion in pancreatic beta cells. Diabetologia 2003; 46:1297-312. [PMID: 13680127 DOI: 10.1007/s00125-003-1207-4] [Citation(s) in RCA: 185] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/14/2003] [Revised: 07/14/2003] [Indexed: 01/16/2023]
Abstract
Increases in glucose or fatty acids affect metabolism via changes in long-chain acyl-CoA formation and chronically elevated fatty acids increase total cellular CoA. Understanding the response of pancreatic beta cells to increased amounts of fuel and the role that altered insulin secretion plays in the development and maintenance of obesity and Type 2 diabetes is important. Data indicate that the activated form of fatty acids acts as an effector molecule in stimulus-secretion coupling. Glucose increases cytosolic long-chain acyl-CoA because it increases the "switch" compound malonyl-CoA that blocks mitochondrial beta-oxidation, thus implementing a shift from fatty acid to glucose oxidation. We present arguments in support of the following: (i) A source of fatty acid either exogenous or endogenous (derived by lipolysis of triglyceride) is necessary to support normal insulin secretion; (ii) a rapid increase of fatty acids potentiates glucose-stimulated secretion by increasing fatty acyl-CoA or complex lipid concentrations that act distally by modulating key enzymes such as protein kinase C or the exocytotic machinery; (iii) a chronic increase of fatty acids enhances basal secretion by the same mechanism, but promotes obesity and a diminished response to stimulatory glucose; (iv) agents which raise cAMP act as incretins, at least in part, by stimulating lipolysis via beta-cell hormone-sensitive lipase activation. Furthermore, increased triglyceride stores can give higher rates of lipolysis and thus influence both basal and stimulated insulin secretion. These points highlight the important roles of NEFA, LC-CoA, and their esterified derivatives in affecting insulin secretion in both normal and pathological states.
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Affiliation(s)
- G C Yaney
- Boston University School of Medicine, Obesity Research Center, 650 Albany Street, Boston, MA 02118, USA
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22
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Lindschau C, Quass P, Menne J, Güler F, Fiebeler A, Leitges M, Luft FC, Haller H. Glucose-induced TGF-beta1 and TGF-beta receptor-1 expression in vascular smooth muscle cells is mediated by protein kinase C-alpha. Hypertension 2003; 42:335-41. [PMID: 12939231 DOI: 10.1161/01.hyp.0000087839.72582.dd] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Sclerosis and increased matrix expression in diabetes are mediated by glucose-induced transforming growth factor (TGF)-beta1 expression. The intracellular effects of high glucose occur at least in part by way of protein kinase C (PKC). We previously described a role for PKC-alpha in glucose-induced permeability. We now investigated the hypothesis that glucose-induced expression of TGF-beta1 and its receptors (TGF-beta-R1 and -R2) are mediated by activation of this PKC isoform. TGF-beta1 and TGF-beta-R expressions were determined in vascular smooth muscle cells (VSMCs) by immunocytochemistry and Western blotting. PKC isoforms were assessed by confocal microscopy. PKC isoforms were inhibited with antisense oligodeoxynucleotides. PKC-alpha was upregulated by overexpression or microinjection. High glucose (20 mmol/L) increased VSMC TGF-beta1 and TGF-beta-R1 expression but not TGF-beta-R2 expression. PKC inhibitors and specific PKC-alpha downregulation by antisense treatment prevented this effect, whereas antisense treatment against PKC-beta, -epsilon, and -zeta had no influence. PKC-alpha overexpression increased TGF-beta1 and TGF-beta-R1 expression but not TGF-beta-R2 expression. PKC-alpha microinjection into individual VSMCs also increased TGF-beta1 and TGF-beta-R immunofluorescence. Last, VSMCs from PKC-alpha-deficient mice did not respond to high glucose compared with VSMCs from wild-type mice. We propose that high glucose-induced TGF-beta1 and TGF-beta-R1 expression is mediated by PKC-alpha. Our findings suggest an autocrine feedback mechanism and a possible role for PKC-alpha in diabetic vascular disease.
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MESH Headings
- Activin Receptors, Type I/genetics
- Activin Receptors, Type I/metabolism
- Animals
- Blotting, Western
- Cells, Cultured
- Dose-Response Relationship, Drug
- Enzyme Inhibitors/pharmacology
- Fluorescent Antibody Technique
- Gene Expression Regulation/drug effects
- Glucose/pharmacology
- Green Fluorescent Proteins
- Luminescent Proteins/genetics
- Luminescent Proteins/metabolism
- Microscopy, Confocal
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/drug effects
- Muscle, Smooth, Vascular/metabolism
- Naphthalenes/pharmacology
- Oligonucleotides, Antisense/genetics
- Oligonucleotides, Antisense/pharmacology
- Protein Kinase C/antagonists & inhibitors
- Protein Kinase C/genetics
- Protein Kinase C/metabolism
- Protein Kinase C-alpha
- Protein Serine-Threonine Kinases
- Rats
- Receptor, Transforming Growth Factor-beta Type I
- Receptors, Transforming Growth Factor beta/genetics
- Receptors, Transforming Growth Factor beta/metabolism
- Recombinant Fusion Proteins/genetics
- Recombinant Fusion Proteins/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Staurosporine/pharmacology
- Time Factors
- Transforming Growth Factor beta/genetics
- Transforming Growth Factor beta/metabolism
- Transforming Growth Factor beta1
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Ferreira F, Filiputti E, Arantes VC, Stoppiglia LF, Araújo EP, Delghingaro-Augusto V, Latorraca MQ, Toyama MH, Boschero AC, Carneiro EM. Decreased cholinergic stimulation of insulin secretion by islets from rats fed a low protein diet is associated with reduced protein kinase calpha expression. J Nutr 2003; 133:695-9. [PMID: 12612139 DOI: 10.1093/jn/133.3.695] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Undernutrition has been shown to affect the autonomic nervous system, leading to permanent alterations in insulin secretion. To understand these interactions better, we investigated the effects of carbamylcholine (CCh) and phorbol 12-myristate 13-acetate (PMA) on insulin secretion in pancreatic islets from rats fed a normal (17%; NP) or low (6%; LP) protein diet for 8 wk. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 8.3 mmol glucose/L, with or without PMA (400 nmol/L) and CCh. Increasing concentrations of CCh (0.1-1000 micro mol/L) dose dependently increased insulin secretion by islets from both groups of rats. However, insulin secretion by islets from rats fed the NP diet was significantly higher than that of rats fed the LP diet, and the dose-response curve to CCh was shifted to the right in islets from rats fed LP with a 50% effective concentration (EC(50)) of 2.15 +/- 0.7 and 4.64 +/- 0.1 micro mol CCh/L in islets of rats fed NP and LP diets, respectively (P < 0.05). PMA-induced insulin secretion was higher in islets of rats fed NP compared with those fed LP. Western blotting revealed that the protein kinase (PK)Calpha and phospholipase (PL)Cbeta(1) contents of islets of rats fed LP were 30% lower than those of islets of rats fed NP (P < 0.05). In addition, PKCalpha mRNA expression was reduced by 50% in islets from rats fed LP. In conclusion, a reduced expression of PKCalpha and PLCbeta(1) may be involved in the decreased insulin secretion by islets from LP rats after stimulation with CCh and PMA.
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Affiliation(s)
- Fabiano Ferreira
- Departamento de Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), SP, Brasil
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24
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Gonelle-Gispert C, Costa M, Takahashi M, Sadoul K, Halban P. Phosphorylation of SNAP-25 on serine-187 is induced by secretagogues in insulin-secreting cells, but is not correlated with insulin secretion. Biochem J 2002; 368:223-32. [PMID: 12164783 PMCID: PMC1222969 DOI: 10.1042/bj20020896] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2002] [Revised: 07/29/2002] [Accepted: 08/06/2002] [Indexed: 11/17/2022]
Abstract
The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N -ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is implicated in regulated insulin secretion. In pheochromocytoma PC12 cells, SNAP-25 is phosphorylated at Ser(187), which lies in a region that is important for its function. The aims of the present study were to determine whether SNAP-25 is phosphorylated at Ser(187) in insulin-secreting cells and, if so, whether this is important for regulated insulin secretion. The major findings are: (i) SNAP-25 is rapidly and reversibly phosphorylated on Ser(187) in both rat insulinoma INS-1 cells and rat islets in response to the phorbol ester, PMA; (ii) less than 35% of SNAP-25 in INS-1 cells is phosphorylated in response to PMA, and phosphorylation is limited to plasma-membrane-associated SNAP-25; (iii) both SNAP-25 isoforms (a and b) are phosphorylated, with 1.8-fold greater phosphorylation for SNAP-25b in response to PMA; (iv) in rat islets, Ser(187) phosphorylation is stimulated by glucose or carbachol, albeit to a lesser extent than by PMA, but not by cAMP; (v) insulin secretion from botulinum neurotoxin E-treated hamster insulinoma tumour (HIT) cells, transfected with toxin-resistant Ser(187)-->Ala or Ser(187)-->Asp mutant SNAP-25, was similar to that of wild-type HIT cells. Furthermore, in rat islets no correlation was found between the extent of SNAP-25 phosphorylation at Ser(187) in response to secretagogues and stimulation of insulin release; (vi) use of protein kinase C (PKC) inhibitors suggests that glucose stimulates SNAP-25 phosphorylation via conventional and non-conventional PKC isoforms. In summary, although SNAP-25 phosphorylation at Ser(187) occurs in insulin-secreting cells and is mediated by PKC, it does not appear to play a major role in regulated insulin secretion.
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Affiliation(s)
- Carmen Gonelle-Gispert
- Laboratoires de Recherche Louis Jeantet, Centre Médical Universitaire, rue Michel Servet, CH-1211 Geneva 4, Switzerland.
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25
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Yaney GC, Fairbanks JM, Deeney JT, Korchak HM, Tornheim K, Corkey BE. Potentiation of insulin secretion by phorbol esters is mediated by PKC-alpha and nPKC isoforms. Am J Physiol Endocrinol Metab 2002; 283:E880-8. [PMID: 12376314 DOI: 10.1152/ajpendo.00474.2001] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Culturing clonal beta-cells (HIT-T15) overnight in the presence of phorbol ester [phorbol myristate acetate (PMA)] enhanced insulin secretion while causing downregulation of some protein kinase C (PKC) isoforms and most PKC activity. We show here that this enhanced secretion required the retention of PMA in the cell. Hence, it could not be because of long-lived phosphorylation of cellular substrates by the isoforms that were downregulated, namely PKC-alpha, -betaII, and -epsilon, but could be because of the continued activation of the two remaining diacylglycerol-sensitive isoforms delta and mu. The enhanced secretion did not involve changes in glucose metabolism, cell membrane potential, or intracellular Ca2+ handling, suggesting a distal effect. PMA washout caused the loss of the enhanced response, but secretion was then stimulated by acute readdition of PMA or bombesin. The magnitude of this restimulation appeared dependent on the mass of PKC-alpha, which was rapidly resynthesized during PMA washout. Therefore, stimulation of insulin secretion by PMA, and presumably by endogenous diacylglycerol, involves the activation of PKC isoforms delta and/or mu, and also PKC-alpha.
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Affiliation(s)
- Gordon C Yaney
- Obesity Research Center, Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
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26
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Pinton P, Tsuboi T, Ainscow EK, Pozzan T, Rizzuto R, Rutter GA. Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells. J Biol Chem 2002; 277:37702-10. [PMID: 12149258 DOI: 10.1074/jbc.m204478200] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.
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Affiliation(s)
- Paolo Pinton
- Henry Wellcome Signalling Laboratories and the Department of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom
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27
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Milkiewicz P, Roma MG, Elias E, Coleman R. Hepatoprotection with tauroursodeoxycholate and beta muricholate against taurolithocholate induced cholestasis: involvement of signal transduction pathways. Gut 2002; 51:113-9. [PMID: 12077103 PMCID: PMC1773293 DOI: 10.1136/gut.51.1.113] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
BACKGROUND Tauroursodeoxycholate (TUDC) provides partial protection against taurolithocholate (TLC) induced cholestasis, possibly by inducing a signalling cascade activating protein kinase C (PKC). The potential protective effects of beta muricholic acid (beta-MC), another 7-beta-hydroxylated bile salt, have not previously been studied in TLC cholestasis. AIMS To study the effect of beta-MC on TLC induced cholestasis and also to investigate further the effects of agents affecting intracellular signalling, notably DBcAMP (a cell permeable cAMP analogue) and several protein kinase inhibitors. METHODS Functional studies were carried out analysing the proportion of hepatocyte couplets able to accumulate the fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF) into their sealed canalicular vacuole (cVA of CLF assay). RESULTS It was found that both beta-MC and DBcAMP were as effective as TUDC in protecting against TLC induced cholestasis. The PKC inhibitors staurosporin and H7 but not the specific protein kinase A (PKA) inhibitor KT5720 abolished the protective effects of TUDC and beta-MC. BAPTA/AM, a chelator of intracellular Ca(2+), significantly decreased the protective effect of both bile salts, and that of DBcAMP. PKC and PKA inhibitors had no effect on protection with DBcAMP. CONCLUSIONS Beta-MC was as effective as TUDC in protecting against TLC cholestasis. Mobilisation of Ca(2+) and activation of PKC, but not of PKA, are involved in the anticholestatic effect of the two 7-beta-hydroxylated bile salts. The hepatoprotective effects of DBcAMP involved Ca(2+) mobilisation, but not PKC or PKA activation.
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Affiliation(s)
- P Milkiewicz
- School of Biosciences, and Liver and Hepatobiliary Unit, University of Birmingham, Birmingham B17 2TT, UK
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28
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Hennige AM, Fritsche A, Strack V, Weigert C, Mischak H, Borboni P, Renn W, Häring HU, Kellerer M. PKC zeta enhances insulin-like growth factor 1-dependent mitogenic activity in the rat clonal beta cell line RIN 1046-38. Biochem Biophys Res Commun 2002; 290:85-90. [PMID: 11779137 DOI: 10.1006/bbrc.2001.6144] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Protein kinase C seems to be linked to the regulation of insulin secretion as well as mitogenic signaling in pancreatic beta cells. To study the impact of different PKC isoforms on insulin secretion and mitogenic activity we stably overexpressed the PKC isoforms alpha, beta2, epsilon, and zeta in the rat clonal beta cell line RIN 1046-38. Under basal conditions PKC alpha, beta2, epsilon, and zeta were identified mainly in the cytosol. Treatment with the phorbol ester TPA caused translocation of PKC alpha, beta2, and epsilon to the plasma membrane. Glucose- and TPA-dependent increases in insulin release were comparable in all cell lines regardless of whether PKC was overexpressed or not. While PKC isoforms alpha, beta2, and epsilon had no effect on the [(3)H]thymidine incorporation rate, overexpression of PKC zeta specifically increased basal as well as IGF-1-dependent [(3)H]thymidine incorporation. Incubation with the MAP-kinase inhibitor PD98056 abolished this effect. Furthermore, treatment with IGF-1 led to activation of the beta cell-specific transcription factor PDX-1 in RIN 1046-38 cells overexpressing PKC zeta. Our data suggest that PKC zeta is involved in basal as well as IGF-1-dependent mitogenesis in RIN 1046-38 cells, while none of the PKC isoforms tested seem to be related to glucose-stimulated insulin release.
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Affiliation(s)
- Anita M Hennige
- Department of Internal Medicine IV, University of Tübingen, Otfried-Mueller-Strasse 10, D-72076 Tübingen, Germany
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29
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Abstract
Insulin secretion from pancreatic islet beta-cells is a tightly regulated process, under the close control of blood glucose concentrations, and several hormones and neurotransmitters. Defects in glucose-triggered insulin secretion are ultimately responsible for the development of type II diabetes, a condition in which the total beta-cell mass is essentially unaltered, but beta-cells become progressively "glucose blind" and unable to meet the enhanced demand for insulin resulting for peripheral insulin resistance. At present, the mechanisms by which glucose (and other nutrients including certain amino acids) trigger insulin secretion in healthy individuals are understood only in part. It is clear, however, that the metabolism of nutrients, and the generation of intracellular signalling molecules including the products of mitochondrial metabolism, probably play a central role. Closure of ATP-sensitive K+(K(ATP)) channels in the plasma membrane, cell depolarisation, and influx of intracellular Ca2+, then prompt the "first phase" on insulin release. However, recent data indicate that glucose also enhances insulin secretion through mechanisms which do not involve a change in K(ATP) channel activity, and seem likely to underlie the second, sustained phase of glucose-stimulated insulin secretion. In this review, I will discuss recent advances in our understanding of each of these signalling processes.
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Affiliation(s)
- G A Rutter
- Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, UK.
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30
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Gilon P, Henquin JC. Mechanisms and physiological significance of the cholinergic control of pancreatic beta-cell function. Endocr Rev 2001; 22:565-604. [PMID: 11588141 DOI: 10.1210/edrv.22.5.0440] [Citation(s) in RCA: 181] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Acetylcholine (ACh), the major parasympathetic neurotransmitter, is released by intrapancreatic nerve endings during the preabsorptive and absorptive phases of feeding. In beta-cells, ACh binds to muscarinic M(3) receptors and exerts complex effects, which culminate in an increase of glucose (nutrient)-induced insulin secretion. Activation of PLC generates diacylglycerol. Activation of PLA(2) produces arachidonic acid and lysophosphatidylcholine. These phospholipid-derived messengers, particularly diacylglycerol, activate PKC, thereby increasing the efficiency of free cytosolic Ca(2+) concentration ([Ca(2+)](c)) on exocytosis of insulin granules. IP3, also produced by PLC, causes a rapid elevation of [Ca(2+)](c) by mobilizing Ca(2+) from the endoplasmic reticulum; the resulting fall in Ca(2+) in the organelle produces a small capacitative Ca(2+) entry. ACh also depolarizes the plasma membrane of beta-cells by a Na(+)- dependent mechanism. When the plasma membrane is already depolarized by secretagogues such as glucose, this additional depolarization induces a sustained increase in [Ca(2+)](c). Surprisingly, ACh can also inhibit voltage-dependent Ca(2+) channels and stimulate Ca(2+) efflux when [Ca(2+)](c) is elevated. However, under physiological conditions, the net effect of ACh on [Ca(2+)](c) is always positive. The insulinotropic effect of ACh results from two mechanisms: one involves a rise in [Ca(2+)](c) and the other involves a marked, PKC-mediated increase in the efficiency of Ca(2+) on exocytosis. The paper also discusses the mechanisms explaining the glucose dependence of the effects of ACh on insulin release.
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Affiliation(s)
- P Gilon
- Unité d'Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine, B-1200 Brussels, Belgium.
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31
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Zawalich WS, Zawalich KC. Effects of protein kinase C inhibitors on insulin secretory responses from rodent pancreatic islets. Mol Cell Endocrinol 2001; 177:95-105. [PMID: 11377825 DOI: 10.1016/s0303-7207(01)00422-1] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The contribution of protein kinase C (PKC) to the regulation of insulin release from perifused islets was explored using staurosporine or Gö 6976 to inhibit the enzyme. Phorbol 12-myristate 13-acetate (PMA, 500 nM) addition to rat islets resulted in a slowly rising insulin secretory response. While minimally effective alone, the addition of 500 nM forskolin together with PMA resulted in a synergistic secretory response. The conventional protein-kinase-C isoform inhibitor Gö 6976 (1 microM) completely abolished PMA-induced secretion. However, the combination of forskolin plus PMA significantly enhanced secretion from Gö 6976-treated islets. Similar to previous findings made with staurosporine, Gö 6976 (1 microM) enhanced the first phase and reduced the second phase of 20 mM glucose-induced secretion from rat islets. Additional studies were conducted comparing the secretory responses of perifused rat or mouse islets to glucose. Dramatic species differences to the hexose were observed. For example, 35-40 min after the onset of stimulation with 8, 10 or 20 mM glucose insulin release rates from mouse islets averaged 32+/-6, 84+/-27 or 131+/-17 pg/islet per minute, respectively. The responses from rat islets averaged 115+/-28, 561+/-112 or 800+/-46 pg/islet per minute at this time point. Islet insulin stores were comparable in both species. The addition of 5 microM carbachol, 500 nM forskolin or 20 mM KCl to mouse islets together with 20 mM glucose resulted in a dramatic augmentation of insulin output. The responses to carbachol or forskolin, but not KCl, were inhibited by 50 nM staurosporine. However, staurosporine (50 nM) reduced insulin secretion from rat islets stimulated with KCl plus 20 mM glucose. Gö 6976 potentiated 20 mM glucose-induced secretion from mouse islets. These studies demonstrate that 1 microM Gö 6976 completely abolishes PMA-induced release from rat islets and has a modest inhibitory effect on 20 mM glucose-induced secretion. Gö 6976 (1 microM) had no inhibitory effect on 20 mM glucose-induced release from mouse islets. These studies also confirm that staurosporine inhibits both PKC- and PKA-mediated events in islets and this lack of specificity may account for its more pronounced inhibition of release when compared to Gö 6976. Finally, significant species differences to PKC inhibitors exist between mouse and rat islets.
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Affiliation(s)
- W S Zawalich
- Yale University School of Nursing, 100 Church Street South, New Haven, CT 06536-0740, USA.
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32
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Nesher R, Warwar N, Khan A, Efendic S, Cerasi E, Kaiser N. Defective stimulus-secretion coupling in islets of Psammomys obesus, an animal model for type 2 diabetes. Diabetes 2001; 50:308-14. [PMID: 11272141 DOI: 10.2337/diabetes.50.2.308] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Psammomys obesus is a model of type 2 diabetes that displays resistance to insulin and deranged beta-cell response to glucose. We examined the major signaling pathways for insulin release in P. obesus islets. Islets from hyperglycemic animals utilized twice as much glucose as islets from normoglycemic diabetes-prone or diabetes-resistant controls but exhibited similar rates of glucose oxidation. Fractional oxidation of glucose was constant in control islets over a range of concentrations, whereas islets from hyperglycemic P. obesus showed a decline at high glucose. The mitochondrial substrates alpha-ketoisocaproate and monomethyl succinate had no effect on insulin secretion in P. obesus islets. Basal insulin release in islets from diabetes-resistant P. obesus was unaffected by glucagon-like peptide 1 (GLP-1) or forskolin, whereas that of islets of the diabetic line was augmented by the drugs. GLP-1 and forskolin potentiated the insulin response to maximal (11.1 mmol/l) glucose in islets from all groups. The phorbol ester phorbol myristic acid (PMA) potentiated basal insulin release in islets from prediabetic animals, but not those from hyperglycemic or diabetes-resistant P. obesus. At the maximal stimulatory glucose concentration, PMA potentiated insulin response in islets from normoglycemic prediabetic and diabetes-resistant P. obesus but had no effect on islets from hyperglycemic P. obesus. Maintenance of islets from hyperglycemic P. obesus for 18 h in low (3.3 mmol/l) glucose in the presence of diazoxide (375 pmol/l) dramatically improved the insulin response to glucose and restored the responsiveness to PMA. Immunohistochemical analysis indicated that hyperglycemia was associated with reduced expression of alpha-protein kinase C (PKC) and diminished translocation of lambda-PKC. In summary, we found that 1) P. obesus islets have low oxidative capacity, probably resulting in limited ability to generate ATP to initiate and drive the insulin secretion; 2) insulin response potentiated by cyclic AMP-dependent protein kinase is intact in P. obesus islets, and increased sensitivity to GLP-1 or forskolin in the diabetic line may be secondary to increased sensitivity to glucose; and 3) islets of hyperglycemic P. obesus display reduced expression of alpha-PKC and diminished translocation of lambda-PKC associated with impaired response to PMA. We conclude that low beta-cell oxidative capacity coupled with impaired PKC-dependent signaling may contribute to the animals' poor adaptation to a high-energy diet.
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Affiliation(s)
- R Nesher
- Department of Endocrinology and Metabolism, Hebrew University-Hadassah Medical Center, Jerusalem, Israel.
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33
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The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane. Biochem J 2001. [PMID: 10926839 DOI: 10.1042/0264-6021:3500155] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
We have investigated the mechanism responsible for the diffusive component of intestinal glucose absorption, the major route by which glucose is absorbed. In perfused rat jejunum in vivo, absorption was strongly inhibited by phloretin, an inhibitor of GLUT2. The GLUT2 level at the brush-border membrane increased some 2-fold when the luminal glucose concentration was changed from 0 to 100 mM. The phloretin-sensitive or diffusive component of absorption appeared superficially linear and consistent with simple diffusion, but was in fact carrier-mediated and co-operative (n=1.6, [G(1/2)]=56 mM; where [G(1/2)] is the glucose concentration at half V(max)) because of the glucose-induced activation and recruitment of GLUT2 to the brush-border membrane. Diffusive transport by paracellular flow was negligible. The phloretin-insensitive, SGLT1-mediated, component of glucose absorption showed simple saturation kinetics with [G(1/2)]=27 mM: the activation of protein kinase C (PKC) betaII, the isoenzyme of PKC that most probably controls GLUT2 trafficking [Helliwell, Richardson, Affleck and Kellett (2000) Biochem. J. 350, 149-154], also showed simple saturation kinetics, with [G(1/2)]=21 mM. We conclude that the principal route for glucose absorption is by GLUT2-mediated facilitated diffusion across the brush-border membrane, which is up to 3-fold greater than that by SGLT1; the magnitude of the diffusive component at any given glucose concentration correlates with the SGLT1-dependent activation of PKC betaII. The implications of these findings for the assimilation of sugars immediately after a meal are discussed.
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34
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Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C. Biochem J 2001. [PMID: 10926838 DOI: 10.1042/0264-6021:3500149] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Perfusion of rat jejunum in vitro with PMA increased fructose transport by 70% compared with control values and was blocked by the protein kinase C (PKC) inhibitor chelerythrine. The brush-border membrane contained both the fructose transporters GLUT5 and GLUT2; the presence of the latter was confirmed by luminal biotinylation. PMA increased the GLUT2 level 4-fold within minutes, so that the level was comparable with that of the basolateral membrane, but had no effect on GLUT5 level. GLUT2 was functional, accessible to luminal fructose and could be inhibited selectively by phloretin to permit determination of GLUT2- and GLUT5-mediated transport components. The 4-fold increase in GLUT2 level induced by PMA was matched by a 4-fold increase in GLUT2-mediated transport: there was a compensatory fall in the GLUT5-mediated rate. The pattern of dynamic trafficking was seen only for GLUT2, not GLUT5 or SGLT1, implying that GLUT2 trafficks to the brush-border membrane by a different pathway. Trafficking of GLUT2 to the brush-border membrane correlated with activation of PKC betaII, implying that this isoenzyme is likely to control trafficking. Since PKC is activated by endogenous hormones, GLUT2 levels in vivo are 3-4-fold those in vitro; moreover, because PKC is inactivated as soon as intestine is excised, GLUT2 is lost from the brush-border within minutes in vitro. It is therefore difficult to detect GLUT2 in most in vitro preparations and its role in intestinal sugar absorption across the brush-border membrane has accordingly been overlooked.
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35
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Kellett GL, Helliwell PA. The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane. Biochem J 2000; 350 Pt 1:155-62. [PMID: 10926839 PMCID: PMC1221237] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/17/2023]
Abstract
We have investigated the mechanism responsible for the diffusive component of intestinal glucose absorption, the major route by which glucose is absorbed. In perfused rat jejunum in vivo, absorption was strongly inhibited by phloretin, an inhibitor of GLUT2. The GLUT2 level at the brush-border membrane increased some 2-fold when the luminal glucose concentration was changed from 0 to 100 mM. The phloretin-sensitive or diffusive component of absorption appeared superficially linear and consistent with simple diffusion, but was in fact carrier-mediated and co-operative (n=1.6, [G(1/2)]=56 mM; where [G(1/2)] is the glucose concentration at half V(max)) because of the glucose-induced activation and recruitment of GLUT2 to the brush-border membrane. Diffusive transport by paracellular flow was negligible. The phloretin-insensitive, SGLT1-mediated, component of glucose absorption showed simple saturation kinetics with [G(1/2)]=27 mM: the activation of protein kinase C (PKC) betaII, the isoenzyme of PKC that most probably controls GLUT2 trafficking [Helliwell, Richardson, Affleck and Kellett (2000) Biochem. J. 350, 149-154], also showed simple saturation kinetics, with [G(1/2)]=21 mM. We conclude that the principal route for glucose absorption is by GLUT2-mediated facilitated diffusion across the brush-border membrane, which is up to 3-fold greater than that by SGLT1; the magnitude of the diffusive component at any given glucose concentration correlates with the SGLT1-dependent activation of PKC betaII. The implications of these findings for the assimilation of sugars immediately after a meal are discussed.
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Affiliation(s)
- G L Kellett
- Department of Biology, University of York, PO Box 373, York YO10 5YW, U.K.
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36
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Helliwell PA, Richardson M, Affleck J, Kellett GL. Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C. Biochem J 2000; 350 Pt 1:149-54. [PMID: 10926838 PMCID: PMC1221236] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/17/2023]
Abstract
Perfusion of rat jejunum in vitro with PMA increased fructose transport by 70% compared with control values and was blocked by the protein kinase C (PKC) inhibitor chelerythrine. The brush-border membrane contained both the fructose transporters GLUT5 and GLUT2; the presence of the latter was confirmed by luminal biotinylation. PMA increased the GLUT2 level 4-fold within minutes, so that the level was comparable with that of the basolateral membrane, but had no effect on GLUT5 level. GLUT2 was functional, accessible to luminal fructose and could be inhibited selectively by phloretin to permit determination of GLUT2- and GLUT5-mediated transport components. The 4-fold increase in GLUT2 level induced by PMA was matched by a 4-fold increase in GLUT2-mediated transport: there was a compensatory fall in the GLUT5-mediated rate. The pattern of dynamic trafficking was seen only for GLUT2, not GLUT5 or SGLT1, implying that GLUT2 trafficks to the brush-border membrane by a different pathway. Trafficking of GLUT2 to the brush-border membrane correlated with activation of PKC betaII, implying that this isoenzyme is likely to control trafficking. Since PKC is activated by endogenous hormones, GLUT2 levels in vivo are 3-4-fold those in vitro; moreover, because PKC is inactivated as soon as intestine is excised, GLUT2 is lost from the brush-border within minutes in vitro. It is therefore difficult to detect GLUT2 in most in vitro preparations and its role in intestinal sugar absorption across the brush-border membrane has accordingly been overlooked.
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Affiliation(s)
- P A Helliwell
- Department of Biology, University of York, PO Box 373, York YO10 5YW, U.K
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37
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Smallwood JI, Malawista SE. Analysis of the PKC-gamma-related immunocrossreactive region of a novel leukocyte protein gamma-rp. Inflammation 1999; 23:387-410. [PMID: 10443800 DOI: 10.1023/a:1020265701407] [Citation(s) in RCA: 76] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The newly discovered gamma-PKC-related-protein of human leukocytes (gamma-rp) crossreacts with a polyclonal antibody preparation originally designed to be specific for PKC-gamma (gammaMb-Ab). As this antibody is currently the only suitable probe for gamma-rp, we sought to characterize the binding of the two proteins. We determined that the gamma Mg-Ab does not recognize the native form of gamma-rp. However, with denaturing immunoblots of gamma-rp, we found that 1) the crossreactive gamma-rp epitope differs somewhat from that of classic rat brain PKC-gamma, but probably only to the degree of the rat/human PKC species difference; 2) the previously reported doublet bands of gamma-rp represent a single protein with cell-stimulus inducible modifications; 3) antibodies present in the gammaMg-Ab pool bind to two separate sites within the gamma-rp epitope; 4) access to one binding site is conformationally restricted, even after protein denaturation; 5) agonist-induced modification of gamma-rp does not significantly affect the total amount of gamma Mg-Ab that it can bind, but 6) does significantly affect the rate of antibody binding to one site. This investigation defines the appropriate experimental use of our antibody, and the significance of these findings for the future study and cloning of gamma-rp is discussed.
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Affiliation(s)
- J I Smallwood
- Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8031, USA
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Beuers U, Probst I, Soroka C, Boyer JL, Kullak-Ublick GA, Paumgartner G. Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes. Hepatology 1999; 29:477-82. [PMID: 9918925 DOI: 10.1002/hep.510290227] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The protein kinase C (PKC) family of isoenzymes plays a key role in the regulation of hepatocellular secretion. The hydrophobic and cholestatic bile acid, taurolithocholic acid (TLCA), acts as a potent Ca++ agonist in isolated hepatocytes. However, its effect on PKC isoforms has not been elucidated. Here we investigate the effects of TLCA at low micromolar concentrations on the distribution of PKC isoforms and on membrane-associated PKC activity. The distribution of PKC isoforms was determined in isolated rat hepatocytes in short-term culture using Western blotting and immunofluorescence techniques. PKC activity was measured radiochemically. TLCA (10 micromol/L) induced selective translocation of epsilon-PKC by 47.9% +/- 20.5% (P <.02 vs. controls; n = 7), but not of alpha-, delta-, and zeta-PKC to the hepatocellular membranes, whereas the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (1 micromol/L) caused translocation of all mobile isoforms, alpha-, delta-, and epsilon-PKC, as shown by immunoblotting. Immunofluorescence studies demonstrated selective translocation of epsilon-PKC to the canalicular membranes of isolated rat hepatocyte couplets by TLCA (10 micromol/L), but predominant translocation to intracellular and basolateral membranes by PMA (1 micromol/L). Both TLCA (10 micromol/L) and PMA (1 micromol/L) stimulated membrane-bound PKC activity by 60.5% +/- 45. 8% (P <.05 vs. controls; n = 5) and 72.4% +/- 37.2% (P <.05; n = 5), respectively. TLCA at lower concentrations (5 micromol/L) was less effective. Because activation of epsilon-PKC has been associated with impairment of vesicle-mediated targeting and insertion of membrane proteins in secretory cells, it is attractive to speculate that TLCA reduces bile secretory capacity of the liver cell by activation of epsilon-PKC at the canalicular membrane.
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Affiliation(s)
- U Beuers
- Department of Medicine II, Klinikum Grosshadern, University of Munich, Munich, Germany.
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Aizawa T, Komatsu M, Asanuma N, Sato Y, Sharp GW. Glucose action 'beyond ionic events' in the pancreatic beta cell. Trends Pharmacol Sci 1998; 19:496-9. [PMID: 9871411 DOI: 10.1016/s0165-6147(98)01273-5] [Citation(s) in RCA: 73] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
For normal glucose homeostasis, insulin release by the pancreatic beta cell is vital. Until recently, it was thought that glucose-induced ionic events, such as closure of the ATP-sensitive K+ (KATP) channels, membrane depolarization, activation of the L-type voltage-dependent Ca2+ channels, Ca2+ influx and elevation of cytosolic free Ca2+, constitute the main signalling pathway in beta-cell stimulus-secretion coupling. However, since the discovery of 'non-ionic' glucose actions in the beta cell by the Aizawa and Henquin laboratories in 1991, data have accumulated that strongly indicate the physiological relevance of this signalling pathway. In this review, Toru Aizawa and colleagues discuss how the KATP channel-Ca2+ hypothesis was formulated, what was overlooked in the hypothesis, and then provide a comprehensive view of stimulus-secretion coupling in the beta cell, with an emphasis on non-ionic glucose actions.
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Affiliation(s)
- T Aizawa
- Department of Geriatrics, Endocrinology and Metabolism, Shinshu University School of Medicine, Naganoken, Japan
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Sjöholm A. Aspects of novel sites of regulation of the insulin stimulus-secretion coupling in normal and diabetic pancreatic islets. Endocrine 1998; 9:1-13. [PMID: 9798725 DOI: 10.1385/endo:9:1:1] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/16/1997] [Accepted: 02/12/1998] [Indexed: 11/11/2022]
Abstract
Noninsulin-dependent diabetes mellitus (NIDDM), a major health care problem in the Western world, is a disease typified by a relative deficiency of insulin, leading to vast derangements in glucose and lipid homeostasis with disastrous vascular complications. Despite immense research efforts aimed at a clear understanding of the etiology of this complex disease, the molecular mechanisms causing the disorder still remain elusive. This article reviews extant data from recent publications implicating novel signal transduction pathways as important regulators of the insulin stimulus-secretion coupling in the pancreatic beta-cell. The significance of nitric oxide and serine/threonine protein phosphatases, and their inactivation by insulin secretagogues, glucose metabolites, ATP, GTP, glutamate, and inositol hexaphosphate in this arena is scrutinized. Additionally, also presented is the growing concept that an important signal for insulin secretion may reside in the inextricable interplay between glucose and lipid metabolism, specifically the generation of malonyl-CoA, which inhibits carnitine palmitoyltransferase 1 with the attendant accumulation of long-chain acyl CoA esters. Moreover, attention is directed towards novel intracellular actions of hypoglycemic sulfonylureas in the beta-cell. Finally, the importance of "lipotoxicity" and aberrations in glucose uptake and metabolism in beta-cell dysfunction is given consideration. Future research efforts should aim at further characterization of effects of second messengers on protein phosphorylation elements in beta-cells. Additionally, long-term regulation by glucose and the diabetic state (e.g., fatty acids and ketones) on beta-cell protein phosphatases, pyruvate dehydrogenase, and carnitine palmitoyltransferase 1 needs to be explored in greater depth. Clearly, the detrimental impact of diabetic hyperlipidemia on beta-cell function has been a relatively neglected area, but futu re pharmacological approaches directed at preventing lipotoxicity may prove beneficial in the treatment of diabetes.
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Affiliation(s)
- A Sjöholm
- Department of Molecular Medicine, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden
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Jones PM, Persaud SJ. Protein kinases, protein phosphorylation, and the regulation of insulin secretion from pancreatic beta-cells. Endocr Rev 1998; 19:429-61. [PMID: 9715374 DOI: 10.1210/edrv.19.4.0339] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Affiliation(s)
- P M Jones
- Biomedical Sciences Division, King's College London, United Kingdom.
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42
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Throckmorton DC, Packer CS, Brophy CM. Protein kinase C activation during Ca2+-independent vascular smooth muscle contraction. J Surg Res 1998; 78:48-53. [PMID: 9733617 DOI: 10.1006/jsre.1998.5368] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The cellular signaling mechanisms that modulate the sustained vascular smooth muscle contractions that occur in vasospasm are not known. We and others have hypothesized that a kinase cascade involving protein kinase C (PKC) modulates sustained vascular smooth muscle contraction. The purpose of this investigation was to develop a model in which the traditional contractile pathways involving myosin light chain phosphorylation are not activated and determine if the PKC pathway is activated under these conditions. The phosphorylation of caldesmon, myosin light chain (MLC20), and the specific PKC substrate, MARCKS (myristoylated, alanine-rich C-kinase substrate) was measured in bovine carotid arterial smoothmuscle (BCASM) stimulated with phorbol 12,13-dibutyrate (PDBu) under Ca2+-containing and Ca2+-free conditions. PDBu stimulation led to increases in caldesmon and MARCKS phosphorylation to the same degree in the presence or absence of Ca2+. PDBu stimulation but did not lead to increases in MLC20 phosphorylation over basal levels in Ca2+-free conditions. Immunoblot analysis of BCASM using PKC isoform-specific antibodies demonstrated the presence of one "Ca2+- dependent" PKC isoform: alpha, and two of the "Ca2+-independent" isoforms: epsilon and zeta. These data suggest that Ca2+-independent isoforms of PKC may play a role in the sustained phase of BCASM contractions through a kinase cascade that involves caldesmon and MARCKS phosphorylation but not MLC20 phosphorylation.
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Affiliation(s)
- D C Throckmorton
- Department of Surgery, Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA
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Zawalich WS, Bonnet-Eymard M, Zawalich KC, Yaney GC. Chronic exposure to TPA depletes PKC alpha and augments Ca-dependent insulin secretion from cultured rat islets. THE AMERICAN JOURNAL OF PHYSIOLOGY 1998; 274:C1388-96. [PMID: 9612227 DOI: 10.1152/ajpcell.1998.274.5.c1388] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The insulin secretory responses of rat islets to glucose (15 mM), 12-O-tetradecanoylphorbol 13-acetate (TPA; 500 nM), and potassium (30 mM) were determined from perifused islets cultured for 22-24 h in CMRL-1066 medium (control cultured) or islets cultured in the additional presence of 500 nM TPA. Islet content of protein kinase C alpha (PKC alpha) and serine and threonine phosphoprotein patterns were also monitored after the culture period. Compared with freshly isolated islets, culturing alone had no adverse effect on the capacity of TPA or 30 mM potassium to stimulate secretion or on the islet content of PKC alpha. In agreement with previous studies, culturing in TPA reduced the islet content of immunoreactive PKC alpha by > 95% and abolished the capacity of the phorbol ester to stimulate secretion during a subsequent dynamic perifusion. Culturing in TPA slightly improved the insulin secretory response to 15 mM glucose compared with control-cultured islets; however, sustained rates of 15 mM glucose-induced secretion from these islets were significantly less than the responses of freshly isolated islets. Islets cultured in TPA responded to 30 mM potassium with a markedly amplified insulin secretory response that was abolished by nitrendipine. Enhanced phosphorylation of several islet proteins was also observed in TPA-cultured islets compared with control-cultured islets. These findings demonstrate that culturing alone impairs glucose-induced secretion, a response that is improved but still subnormal compared with freshly isolated islet responses, if TPA is included in the culture medium. Sustained phosphorylation of several islet proteins in TPA-cultured islets may account, at least in part, for augmented calcium-dependent secretion.
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Affiliation(s)
- W S Zawalich
- Yale University School of Nursing, New Haven, Connecticut 06536-0740, USA
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Smallwood JI, Malawista SE. An apparently novel protein of human leukocytes, reactive with an antibody to protein kinase C-gamma, is rapidly modified upon cell activation: initial characterization in neutrophils and their cytoplasts. Inflammation 1998; 22:1-28. [PMID: 9484647 DOI: 10.1023/a:1022390406225] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
On immunoblots of human neutrophil cytoplasts (U-CYT), a previously undescribed 97 kDa protein was revealed by intense and selective reaction with an antibody that was initially raised to recognize PKC-gamma. Denoted "gamma-rp" for gamma-related protein, this acidic cytosolic protein somewhat resembled the classic forms of PKC in several biochemical respects. Appearing as a doublet on low-percentage SDS-PAGE gels, both its mobility and staining pattern were rapidly altered by treatment of U-CYT with either phorbol ester or chemotactic peptide. Whole neutrophil gamma-rp was detectable only after TCA precipitation of intact cells. It was also detectable in human platelets, lymphocytes, and neutrophil-like differentiated HL60 cells, but not in fibroblasts, erythrocytes, monocytes, or monocyte-like differentiated HL60 cells. Our data suggest that gamma-rp merits further study as a potential participant in cellular activation, and as a possible structural or functional relative of PKC.
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Affiliation(s)
- J I Smallwood
- Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8031, USA
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Love JA, Richards NW, Owyang C, Dawson DC. Muscarinic modulation of voltage-dependent Ca2+ channels in insulin-secreting HIT-T15 cells. THE AMERICAN JOURNAL OF PHYSIOLOGY 1998; 274:G397-405. [PMID: 9486195 DOI: 10.1152/ajpgi.1998.274.2.g397] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Potentiation of insulin secretion from pancreatic beta-cells by acetylcholine requires ongoing cyclic electrical activity initiated by other depolarizing secretagogues. Patch-clamp recordings in glucose-free solutions were made from the clonal beta-cell line HIT-T15 to determine whether the muscarinic agonist bethanechol (BCh) modulated voltage-dependent Ca2+ channels independent of effects on membrane potential. Only high-threshold, dihydropyridine-sensitive (L-type) Ca2+ channels with a mean conductance of 26 pS were observed in cell-attached patches. BCh (100 microM) caused a two- to threefold increase in both fractional open time and mean current of single Ca2+ channels. These changes resulted from a 44% decrease in the longer of two apparent mean closed times and a 25% increase in the mean open time. Similar BCh-stimulated increases in macroscopic Ca2+ currents were recorded in whole cell, perforated-patch recordings. The role of protein kinase C (PKC) in the muscarinic activation of Ca2+ channels was tested using a variety of PKC activators and inhibitors. Acute application of either the active phorbol ester phorbol 12-myristate 13-acetate (PMA) or the membrane-permeable diacylglycerol analog 1,2-didecanoyl-rac-glycerol mimicked the effects of BCh, whereas an inactive phorbol (4 alpha) had no effect. Depletion of PKC activity by chronic exposure to PMA or acute application of the PKC inhibitor staurosporine greatly reduced or abolished muscarinic activation of Ca2+ channels. These results are consistent with muscarinic activation of L-type, voltage-dependent Ca2+ channels mediated in large part by PKC.
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Affiliation(s)
- J A Love
- Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson 39216, USA
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Abstract
We have previously identified expression of multiple protein kinase C (PKC) isoforms in insulinoma-derived beta-cells and whole islets. Both PKC delta and PKC alpha appear to be the more abundantly expressed isoforms. In this report we studied the effects of arachidonic acid (AA) on the subcellular distribution of PKC alpha and PKC delta. AA has been reported to activate both PKC alpha and PKC delta and it is thought to be an important second messenger in beta-cells. Here we report that AA interacted with and altered beta-cell pools of PKC delta preferentially over PKC alpha. AA (100 microM) over the course of 45 min reduced cytosolic levels of PKC delta (to 40 +/- 15%, compared to time zero control) leaving membrane- and cytoskeleton-associated levels near control levels. Analysis of whole cell homogenates showed a slight down-regulation of PKC delta indicating proteolysis. The down-regulation of cytosolic PKC delta appeared to be isoform specific since cytosolic PKC alpha remained at control levels over the time course. The response was dose-dependent and negligible at concentrations below 30 microM and occurred, at least partially, in the cytosolic compartment of the cell. Indomethacin also down-regulated cytosolic PKC delta preferentially over PKC alpha possibly through accumulation of AA. These findings suggest that cytosolic PKC delta may be a downstream target of this beta-cell second messenger.
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Affiliation(s)
- K L Knutson
- Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens 30602, USA
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48
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Zawalich WS, Zawalich KC. Regulation of insulin secretion by phospholipase C. THE AMERICAN JOURNAL OF PHYSIOLOGY 1996; 271:E409-16. [PMID: 8843732 DOI: 10.1152/ajpendo.1996.271.3.e409] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Biphasic insulin secretion in response to a sustained glucose stimulus occurs when rat or human islets are exposed to high levels of the hexose. A transient burst of hormone secretion is followed by a rising and sustained secretory response that, in the perfused rat pancreas, is 25- to 75-fold greater than prestimulatory insulin release rates. This insulin secretory response is paralleled by a significant five- to sixfold increase in the phospholipase C (PLC)-mediated hydrolysis of islet phosphoinositide (PI) pools by high glucose. In contrast, mouse islets, when stimulated under comparable conditions with high glucose, display a second-phase response that is flat and only slightly (two- to threefold) greater than prestimulatory release rates. The minimal second-phase insulin secretory response to high glucose is accompanied by the minimal activation of PLC in mouse islets as well. However, stimulation of mouse islets with the protein kinase C (PKC) activator tetradecanoyl phorbol acetate (TPA) or the muscarinic agonist carbachol, which significantly activates an isozyme of PLC distinct from that activated by high glucose, induces a rising and sustained second-phase insulin secretory response. When previously exposed to high glucose, both rat and human islets respond to subsequent restimulation with an amplified insulin secretory response. They display priming, sensitization, or time-dependent potentiation. In contrast, mouse islets primed under similar conditions with high glucose fail to display this amplified insulin secretory response on restimulation. Mouse islets can, however, be primed by brief exposure to either TPA or carbachol. Finally, whereas rat islets are desensitized by chronic exposure to high glucose, mouse islet insulin secretory responses are relatively immune to this adverse effect of the hexose. These and other findings are discussed in relationship to the role being played by agonist-induced increases in the PLC-mediated hydrolysis of islet phosphoinositide pools and the activation of PKC in these species-specific insulin secretory response patterns.
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Affiliation(s)
- W S Zawalich
- Yale University School of Nursing, New Haven, Connecticut 06536-0740, USA
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Deeney JT, Cunningham BA, Chheda S, Bokvist K, Juntti-Berggren L, Lam K, Korchak HM, Corkey BE, Berggren PO. Reversible Ca2+-dependent translocation of protein kinase C and glucose-induced insulin release. J Biol Chem 1996; 271:18154-60. [PMID: 8663368 DOI: 10.1074/jbc.271.30.18154] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
It has been reported that protein kinase C (PKC) interacts at multiple sites in beta-cell stimulus-secretion coupling. Nevertheless, there is still controversy concerning the importance of this enzyme in glucose-induced insulin release. The present study was undertaken to clarify whether glucose, directly, or through changes in cytoplasmic free Ca2+ concentration, [Ca2+]i, could promote translocation of PKC from the soluble to the membrane compartment. Whereas glucose, which increases [Ca2+]i, did not affect long-term distribution of PKC activity between soluble and membrane fractions, this distribution was reversibly affected acutely by the Ca2+ concentration in the extraction media. Translocation of PKC to the membrane by incubation of HIT cells for 10 min in the presence of 20 nM phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a 5-fold increase in glucose-induced insulin release. This was prevented by 50 nM concentration of the PKC inhibitor staurosporine, provided that the cells were exposed to the inhibitor before the phorbol ester. Cells pretreated with TPA demonstrated increased insulin secretion in response to glucose for several hours. This time course extended beyond the disappearance of [3H]TPA from the cells, which was complete after 1 h. Activation of PKC increased both average insulin release and the amplitude of oscillations 2-fold, but did not affect oscillation frequency. The stimulatory effect of increased PKC activity on insulin release was not matched by changes in [Ca2+]i. We suggest that stimulation of the pancreatic beta-cell with glucose promotes transient translocation of certain PKC isoforms from the cytoplasm to the plasma membrane as a direct consequence of the increase in [Ca2+]i. Such a translocation may promote phosphorylation of one or several proteins involved in the regulation of the beta-cell stimulus-secretion coupling. This results in potentiation of glucose-induced activation of insulin exocytosis, an effect then not mediated by an increase in [Ca2+]i per se. Hence, pulsatile insulin release can be obtained under conditions where overall [Ca2+]i does not change, challenging the view that oscillations in [Ca2+ ]i are indeed driving the oscillations in hormone release.
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Affiliation(s)
- J T Deeney
- Boston University Medical Center, Diabetes and Metabolism Unit, Boston, Massachusetts 02118, USA
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Florin-Christensen J, Florin-Christensen M, Meinardi E, Calle R. Diversity of roles of protein kinase C alpha in the proliferation of Swiss 3T3 cells. Biochem J 1996; 315 ( Pt 2):513-6. [PMID: 8615822 PMCID: PMC1217225 DOI: 10.1042/bj3150513] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
We examined the role of protein kinase C alpha (PKC alpha ) in the stimulation of DNA synthesis of Swiss 3T3 cells induced by bombesin, platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA). We found that cells in which this kinase had been down-regulated showed a partially abrogated mitogenic response to bombesin. The response to PDGF was unaltered; however, the response to PMA was completely suppressed. The mitogenic effect of maximal doses of bombesin and PMA combined was greater than that of either agent alone, suggesting that bombesin does not fully activate the PKC pathway. Accordingly, bombesin-induced PKC alpha translocation from cytosol to membranes was partial, while that observed with PMA was essentially complete. Moreover, exposure to Ro-31-8220, a PKC inhibitor, had significantly greater effects on the response to PMA than on that to bombesin. Our findings point out different roles that PKC alpha may play in diversely activated cells: while, in the case of PMA, stimulation of this kinase may be necessary and sufficient to induce proliferation, it appears to be necessary only for a full response to bombesin, and redundant among the mechanisms triggered by PDGF.
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