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Uncoupling Proteins: Regulation by IGF-1 and Neuroprotection during Hyperglycemia in Vitro
Stockholm University, Faculty of Science, Department of Neurochemistry.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Diabetic neuropathy is believed to arise due to oxidative stress following hyperglycemic situations. Uncoupling proteins (UCPs) constitute a subgroup of mitochondrial transporter proteins with putative antioxidant properties. By dissipating the proton gradient over the mitochondrial inner membrane, these proteins reduce the mitochondrial inner membrane potential (MMP), and thereby, the mitochondrial production of reactive oxygen species (ROS) is decreased. In this thesis I have examined the regulation of UCP2, UCP3, and UCP4 by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1). I have also investigated the possible involvement of UCP3 in IGF-1-mediated neuroprotection following high glucose treatments. All studies were performed using human neuroblastoma SH-SY5Y cells as an in vitro cell model. The major findings were as follows:

i. Native SH-SY5Y cells expressed UCP2, UCP3, and UCP4.

ii. UCP3 was upregulated by IGF-1 via activation of the IGF-1 receptor. IGF-1 increased UCP3 mRNA and protein levels primarily via activation of the “classical” anti-apoptotic phosphatidyl inositol 3 (PI3)-kinase signaling pathway, as shown by incubation with specific inhibitors of the PI3-kinase and mitogen activated protein (MAP) kinase signaling pathways.

iii. UCP2 and UCP4 protein levels were only marginally or not at all regulated by IGF-1. These UCPs are probably not involved in IGF-1-mediated neuroprotection.

iv. High glucose concentrations reduced the UCP3 protein levels in highly differentiated SH-SY5Y cells. Concomitantly, the MMP and the levels of ROS and glutathione increased, whereas the number of neurites per cell was reduced. This supports an antioxidant and neuroprotective role of UCP3

v. IGF-1 prevented the glucose-induced reduction in UCP3 protein levels. In parallel, the effects on MMP, levels of ROS and glutathione, and number of neurites per cell were abolished or significantly reduced. These data suggest that UCP3 is involved in IGF-1-mediated neuroprotection.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi , 2004. , 64 p.
Keyword [en]
uncoupling protein, oxidative stress, diabetes, neuropathy, SH-SY5Y
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:su:diva-121ISBN: 91-7265-826-6 (print)OAI: oai:DiVA.org:su-121DiVA: diva2:189597
Public defence
2004-05-19, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2004-04-28 Created: 2004-04-28 Last updated: 2010-11-19Bibliographically approved
List of papers
1. Insulin-like growth factor type 1 up-regulates uncoupling protein 3
Open this publication in new window or tab >>Insulin-like growth factor type 1 up-regulates uncoupling protein 3
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2001 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 287, no 5, 1105-1111 p.Article in journal (Refereed) Published
Abstract [en]

In this study the expression of uncoupling protein 3 (UCP3) and its regulation by insulin-like growth factor 1 (IGF-I) and insulin in human neuroblastoma SH-SY5Y cells were characterized. Reverse transcriptase-PCR, Western blot, and immunofluorescence analysis showed that SH-SY5Y cells express UCP3 natively. IGF-I induced a time- and concentration-dependent induction of UCP3 protein reaching a twofold expression after 72 h with 10 nM IGF-I. Extremely high insulin concentrations (860 nM) and 10 nM trIGF-I, a truncated form of IGF-I with the same affinity for the IGF-I receptor as the full-length IGF-I, but with lower activity on the insulin receptor, also upregulated UCP3. We conclude that SH-SY5Y cells express UCP3 natively and that the expression is regulated by IGF-I via the IGF-I receptor.

Keyword
uncoupling protein, IGF-I, SH-SY5Y, insulin, diabetes
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-22832 (URN)10.1006/bbrc.2001.5702 (DOI)
Note
Part of urn:nbn:se:su:diva-121Available from: 2004-04-28 Created: 2004-04-28 Last updated: 2015-03-09Bibliographically approved
2. Signalling pathways for insulin-like growth factor type 1-mediated expression of uncoupling protein 3
Open this publication in new window or tab >>Signalling pathways for insulin-like growth factor type 1-mediated expression of uncoupling protein 3
2004 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 88, no 2, 462-468 p.Article in journal (Refereed) Published
Abstract [en]

Uncoupling protein 3 (UCP3) is a mitochondrial protein with antioxidant properties and its regulation by factors promoting cell-survival may be important for protection of, for instance, neurons in states of oxidative stress. In the present study, we investigated regulatory pathways for UCP3 expression mediated by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1) in human neuroblastoma SH-SY5Y cells. Northern blot analysis and RT-PCR showed that treatment with 10 nm IGF-1 increased the UCP3 mRNA levels 2.5-fold after 5 h. Co-incubation with the phosphatidylinositol 3 (PI3)-kinase inhibitor LY294002 prohibited IGF-1-mediated induction of both UCP3 mRNA and protein in a concentration-dependent manner, with a complete blockage at 1 μm, as shown by RT-PCR and western blot analyses. The mitogen-activated protein (MAP) kinase kinase 1 (MKK1 or MEK) inhibitor PD98059 also decreased the UCP3 mRNA expression at 10 μm, however, this concentration only partly inhibited the protein expression. We conclude that IGF-1 enhanced UCP3 expression at transcriptional level, primarily through the PI3-kinase-dependent pathway and partly through the MAP kinase pathway.

Keyword
IGF-1, MAP kinase, oxidative stress, PI3-kinase, SH-SY5Y, uncoupling protein
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-22833 (URN)10.1046/j.1471-4159.2003.02162.x (DOI)
Note
Part of urn:nbn:se:su:diva-121Available from: 2004-04-28 Created: 2004-04-28 Last updated: 2015-03-09Bibliographically approved
3. Expression of uncoupling protein 2 and 4 in human neuroblastoma SH-SY5Y cells
Open this publication in new window or tab >>Expression of uncoupling protein 2 and 4 in human neuroblastoma SH-SY5Y cells
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-22834 (URN)
Note
Part of urn:nbn:se:su:diva-121Available from: 2004-04-28 Created: 2004-04-28 Last updated: 2010-01-13Bibliographically approved
4. Insulin-like growth factor type 1 prevents hyperglycemia-induced uncoupling protein 3 down-regulation and oxidative stress
Open this publication in new window or tab >>Insulin-like growth factor type 1 prevents hyperglycemia-induced uncoupling protein 3 down-regulation and oxidative stress
Show others...
2004 (English)In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 77, no 2, 285-291 p.Article in journal (Refereed) Published
Abstract [en]

Uncoupling proteins (UCPs) have been reported to decrease the mitochondrial production of reactive oxygen species (ROS) by lowering the mitochondrial inner membrane potential (MMP). We have previously shown that UCP3 expression is positively regulated by insulin-like growth factor-1 (IGF-1). The aim of this study was to investigate the role of UCPs in IGF-1-mediated protection from hyperglycemia-induced oxidative stress and neurodegeneration. Human neuroblastoma SH-SY5Y cells were differentiated with retinoic acid for 6 days, after which exposure to 8, 30, or 60 mM glucose with or without 10 nM IGF-1 was started. After 48-72 hr, the number of neurites per cell, UCP3 protein expression, MMP, and intracellular levels of ROS and total glutathione were examined. These studies showed that glucose concentration-dependently reduced the number of neurites per cell, with a 50% reduction at 60 mM. In parallel, the UCP3 protein expression was down-regulated, and the MMP was raised 3.5-fold, compared with those in cells incubated with 8 mM glucose. Also, the ROS levels were increased, showing a twofold maximum at 60 mM glucose. This was accompanied by a twofold elevation of total glutathione levels, confirming an altered cellular redox state. IGF-1 treatment prevented the glucose-induced neurite degeneration and UCP3 down-regulation. Furthermore, the MMP and the intracellular levels of ROS and glutathione were normalized to those of control cells. These data indicate that IGF-1 may protect from hyperglycemia-induced oxidative stress and neuronal injuries by regulating MMP, possibly by the involvement of UCP3.

Keyword
diabetes, neuropathy, uncoupling protein, ROS, SH-SY5Y
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:su:diva-20496 (URN)10.1002/jnr.20142 (DOI)15211595 (PubMedID)
Available from: 2007-11-26 Created: 2007-11-26 Last updated: 2015-03-09Bibliographically approved

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