Uppsala university: Faculty of Medicine: Department of medical cell biology: Research: Nils Welsh
Pancreatic b-cell Research
Project 1: Efficient transduction of islet cells
In this project we compare the efficency and safety of different adeno-, lenti- and AAV vectors for transduction of islet cells in vitro and in situ with the purpose to find the optimal gene delivery method for islet transduction purposes. The Figure below shows that GFP-expressing vectors reach only the outer cells of an intact islet when added in vitro (left panel), whereas using an in situ perfusion based protocol also centrally located cells are transduced.
Selected references:
- Saldeen J, Curiel D, A. Andersson, Eizirik DL, Strandell E, Buschard K and Welsh N. Efficient gene transfer to human pancreatic islets in vitro using adenovirus/polylysine/DNA-conjugates or cationic liposomes. Diabetes, 45: 1197-1203 [1996].
- Barbu AR, Akusjarvi G, Welsh N. Adenoviral-mediated transduction of human pancreatic islets: importance of adenoviral genome for cell viability and association with a deficient antiviral response. Endocrinology. 146:2406-14 (2005)
- Hagerkvist R, Mokhtari D, Myers JW, Tengholm A, Welsh N. siRNA Produced by Recombinant Dicer Mediates Efficient Gene Silencing in Islet Cells. Ann N Y Acad Sci. 1040: 114-22 (2005)
- Barbu A and Welsh N. Lipofection of insulin producing cells: Methodological improvements. J Liposome Res 2007;17(2):49-62.
- Barbu AR, Bodin B, Welsh M, Jansson L, Welsh N. A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans. Diabetologia. 2006 Oct;49(10):2388-91.
Project 2: To genetically modify beta-cells so that they are not destroyed by transplantation-induced stress or immune system-induced autoimmune destruction
In panels A-D insulin producing cells were transfected with a control vector and in panels E-H with a vector that promotes overexpression of the anti-apoptotic protein Bcl-2. In panels B, D, E and H cells were stained with bisbenzimide, which stains living cells blue, and propidium iodide, which stains dead cells pink. In panels A, C, F and G cells were stained with JC-1, which is a marker for a high mitochondrial membrane potential (m). Panels C, D, G and H are cells treated for 24 hours with a cytokine mixture. The figure shows that control cells die by apoptosis (a) and necrosis (n) in response to cytokines and that this is preceded by a loss of the mitochondrial membrane potential (m). In cells tranduced to overexpress Bcl-2, however, there is neither loss of mitochondrial membrane potential nor increased cell death. Other gene products that may influence beta-cell survival in diabetes and that are presently investigated are NF-kappaB, MIF-1, TGF-beta and FasL.
Selected references:
- Welsh N, Bendtzen K and Welsh M. Expression of an an insulin/interleukin-1 receptor antagonist hybrid gene in insulin producing cell lines (HIT-T15 and NIT-1) confers resistence against interleukin-1 induced nitric oxide production. J. Clin. Invest., 95, 1717-1722 [1995]
- Saldeen J, Sandler S, Bendtzen K and Welsh N. Liposome-mediated transfer of IL-1 receptor antagonist gene to dispersed islet cells does not prevent recurrence of disease in syngeneically transplanted NOD mice. Cytokine 12: (4) 405-408 (2000)
- Barbu A, Welsh N, Saldeen J. Cytokine-induced cell death is preceded by disruption of the mitochondrial transmemebrane potential (DFm) in RINm5F cells:Prevention by Bcl-2. Mol Cell Endocrinol. 190, 75-82 (2002)
- Welsh N, Makeeva N, Welsh M. Overexpression of the Shb SH2 domain-protein leads to altered signaling through the IRS-1 and IRS-2 proteins with consequences for phosphoinositide metabolism in insulin producing cells. Mol. Med. 8: 695-704 (2002).
- Barbu A, Akusjärvi G, Welsh N. Adenoviral induced islet cell cytotoxicity is not counteracted by Bcl-2 overexpression. Mol. Med. 8(11): 733-741 (2002)
Project 3: Role of tyrosine kinases in beta-cell apoptosis
Tyrosine kinases seem to control beta-cell death and the tyrosine kinase inhibitor (TKI) Gleevec conteracts diabetes in both streptozotocin-injected mice (Figure below) and in NOD mice. It is the aim of this project to elucidate the mechanisms by which tyrosine kinases control beta-cell death.
Selected references:
- Hagerkvist R, Makeeva N, Elliman S, Welsh N. Imatinib mesylate (Gleevec) protects against streptozotocin-induced diabetes and islet cell death in vitro. Cell Biol Int. 2006 Dec;30(12):1013-7
- Hagerkvist R, Sandler S, Mokhtari D, Welsh N. Amelioration of diabetes by imatinib mesylate (Gleevec): role of beta-cell NF-kappaB activation and anti-apoptotic preconditioning. FASEB J. 2007 Feb;21(2):618-28
- Hagerkvist R, Mokhtari D, Lindholm C, Farnebo F, Mostoslavsky G, Mulligan RC, Welsh N, Welsh M. Consequences of Shb and c-Abl interactions for cell death in response to various stress stimuli.Exp Cell Res. 2007 Jan 15;313(2):284-91
- Robert Hägerkvist, Leif Jansson and Nils Welsh. Imatinib mesylate improves insulin sensitivity and glucose disposal rates in high-fat diet fed rats, Clinical Science, (Lond). 2008 Jan;114(1):65-71.
Project 4: Role of p38 and JNK in beta-cell apoptosis
We have observed that both the two stress-activated MAP kinases p38 and JNK are activated in insulin producing cells in response to cytokines and nitric oxide. Furthermore, they seem to participate in beta-cell death as p38 down-regulation results in partial alleviation of the cytokine/nitric oxide-induced effect. Therefore, it is of great importance to better understand the mechanisms by which p38 and JNK are activated, and how these MAP kinases act in insulin producing cells. The figure below shows human islet cells treated with control (GL3) or Tab1-specific siRNA. Tab1 is a p38 activating protein and down-regulation of Tab1 results in partial protection against cytokines.
Selected references:
- Welsh N. Interleukin-1b induced ceramide and diacylglycerol generation leads to activation of the Jun kinase and the transcription factor ATF2 in the insulin-producing cell line RINm5F. J. Biol. Chem, 271:8307-8312, [1996]
- Saldeen J, Lee JC, Welsh N. Requirement of enhanced p38 mitogen-activated protein kinase activity in cytokine-induced apoptosis in rat islet cells in vitro. Biochemical Pharmacology, 61, 1561-1569 (2001)
- Saldeen J, Welsh N. P38 MAPK inhibits JNK2 and mediates cytokine-activated iNOS induction and apoptosis independently of NF-kB translocation in insulin producing cells. European Cytokine Network 15: 47-52 (2004)
- Welsh N, Cnop M, Kharroubi I, Bugliani M, Marchetti P, Eizirik DL. Is there a role for islet-produced interleukin-1 in the deleterious effects of high glucose to human pancreatic islets? Diabetes 54, 3238-3244 (2005).
- Cnop M, Welsh N, Jonas JC, Jörns A, Lenzen S, Eizirik DL. Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes. 2005 Dec;54 Suppl 2:S97-107. Review.
- Makeeva N, Myers JW, Welsh N. Role of MKK3 and p38 MAPK in cytokine-induced death of insulin producing cells. Biochemical Journal 393, 129-139 (2006)
- Makeeva N, Roomans G and Welsh N. Role of TAB1 in nitric oxide-induced p38 activation in insulin-producing cells. Int J Biol Sci. 2006 Nov 25;3(2):71-6.
- Mokhtari D, Myers JW, Welsh N. The MAPK kinase kinase-1 is essential for stress-induced pancreatic islet cell death. Endocrinology. 2008 Feb 28; [Epub ahead of print]
Control of insulin mRNA stability by pyrimidine tract binding protein (PTB):
Most attempts to understand why insulin mRNA levels are decreased in diabetes have assumed a lowered transcription of the insulin gene. However, we have recently observed that insulin mRNA levels are mainly controlled by post-transcriptional mechanisms and that the 55 kDa pyrimidine tract binding protein (PTB) binds to the 3'-UTR of insulin mRNA. Hypoxia, glucose or mTOR stimulated this binding, and mutation of the core-binding site resulted in reporter mRNA destabilization. The over-all aim of this project is to understand how glucose regulates PTB activity in the control of insulin mRNA stability. This project might generate novel knowledge on the mechanisms behind decreased insulin production in certain types of diabetes.
Selected references:
- Tillmar L, Carlsson C. Welsh N. Control of insulin mRNA stability in rat pancreatic islets: Regulatory role of a 3'-UTR pyrimidine-rich sequence. J. Biol. Chem. 277: 1099-1106 (2002)
- Tillmar L, Welsh N. Hypoxia may increase insulin mRNA contents by stimulating the binding of polypyrimidine tract binding protein to the 3'-UTR of insulin mRNA. Mol. Med. 8:263-272 (2002)
- Tillmar L, Welsh N. Glucose-stimulated binding of polypyrimidine-tract binding protein to the 3’UTR of insulin mRNA is inhibited by rapamycin. Mol. Cell. Biochem. 260: 85-90 (2004)
- Tillmar L, Welsh N. Islet expression of the orphan endothelial cell tyrosine kinase receptor 1 is increased in response to hypoxia in vitro. Journal of the Pancreas. 5: 81-91 (2004)
- Fred R and Welsh N. Increased expression of polypyrimidine tract binding protein in insulin-producing bTC-6 cells results in higher insulin mRNA levels. Biochem. Biophys. Res. Commun. 328: 38-42 (2005)
- Fred RG, Tillmar L, Welsh N. The role of PTB in insulin mRNA stability control. Curr Diabetes Rev. 2006 Aug;2(3):363-6. Review.
Study group members
Nils Welsh - Professor
Andreea Barbu – Post-doc
Darish Mokthari – PhD-student
Rickard Fred – PhD-student
Xuan Wang - Student
Facilities
Cell culture lab
Virus vector lab
Islet isolation lab
Molecular biology lab
Real-time PCR
Flow cytometry and cell sorting
Three laser confocal microscopy
Kodak 4000MM imaging unit
Access to full animal department with facilities for islet transplantation and clamping
Access to human islets Access to electron microscopy and gene array (MPSS) facilities