[show abstract][hide abstract] ABSTRACT: Hyperglycemia in patients with type 2 diabetes causes multiple neuronal complications, e.g., diabetic polyneuropathy, cognitive decline, and embryonic neural crest defects due to increased apoptosis. Possible mechanisms of neuronal response to increased glucose burden are still a matter of debate. Insulin and insulin-like growth factor-1 (IGF-1) receptor signaling inhibits glucose-induced caspase-3 activation and apoptotic cell death. The insulin receptor substrates (IRS) are intracellular adapter proteins mediating insulin's and IGF-1's intracellular effects. Even though all IRS proteins have similar function and structure, recent data suggest different actions of IRS-1 and IRS-2 in mediating their anti-apoptotic effects in glucose neurotoxicity. We therefore investigated the role of IRS-1/-2 in glucose-induced caspase-3 activation using human neuroblastoma cells. Overexpression of IRS-1 or IRS-2 caused complete resistance to glucose-induced caspase-3 cleavage. Inhibition of PI3-kinase reversed this protective effect of IRS-1 or IRS-2. However, MAP-kinases inhibition had only minor impact. IRS overexpression increased MnSOD abundance as well as BAD phosphorylation while Bim and BAX levels remained unchanged. Since Akt promotes cell survival at least partially via phosphorylation and inhibition of downstream forkhead box-O (FoxO) transcription factors, we generated neuroblastoma cells stably overexpressing a dominant negative mutant of FoxO1 mimicking activation of the insulin/IGF-1 pathway on FoxO-mediated transcription. Using these cells we showed that FoxO1 is not involved in neuronal protection mediated by increased IRS-1/-2 expression. Thus, overexpression of both IRS-1 and IRS-2 induces complete resistance to glucose-induced caspase-3 activation via PI3-kinase mediated BAD phosphorylation and MnSOD expression independent of FoxO1.
Biochimica et Biophysica Acta 02/2011; 1812(5):573-80. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Since neuronal insulin receptor substrate 2 (IRS2)-mediated signals coordinate key processes in rodent physiology such as food intake, fertility, longevity, and aging-related behavior, we analyzed the mechanisms of neuronal IRS2 expression in neuroblastoma (SHSY5Y) and hypothalamic (GT1-7) cell lines. Using dual luciferase reporter assays and IRS2 promoter deletion constructs, we identified a regulatory cassette within the IRS2 promoter between -779 and -679 bp from the translational start which is responsible for approximately 50% of neuronal IRS2 promoter activity. Chromatin immunoprecipitation assays and electromobility shift assay revealed four overlapping ZBP89/specificity protein 1 (SP1) binding sites which alternatively bind to ZBP89 (ZNF148 as listed in the HUGO Database) or SP1. Activation of this cassette is inhibited by phosphoinositide-3-kinase (PI3K) via increased ZBP89 binding to the promoter. Serum starvation caused increased SP1 binding at one specific SP1 site and decreased binding to another, proving a regulatory interaction between the different binding sites within this promoter cassette to tightly control IRS2 expression. Mutants containing all the possible combinations of one, two, three, or all the four SP1 binding sites of the IRS2 promoter revealed that SP1 binding to one particular site is most important for promoter activation. Stable downregulation of ZBP89 using siRNA substantially increased IRS2 mRNA and protein expression. Thus, alternative binding of ZBP89 or SP1 to the described region in the IRS2 promoter regulates neuronal IRS2 expression in a PI3K-dependent manner.
Journal of Endocrinology 10/2009; 204(2):199-208. · 4.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent studies have discovered changes in the insulin-/IGF1 signaling affecting glucose metabolism and the molecular pathogenesis of human hepatocellular cancer. Insulin/IGF1 receptor mediates its intracellular effects by recruitment of one out of the four different insulin receptor substrates (IRS). To investigate mechanisms of IRS2 expression, we analyzed transcriptional regulation of IRS2 in human HepG2 cells. We identified a region 688 bp upstream of the translation start codon responsible for approximately 90% of basal human IRS2 promoter activity in HepG2 cells, and confirmed binding of specificity protein 1 (also called Sp1 transcription factor, SP1) and nuclear factor 1 (NFI) in this region. Mutation of both SP1 and NFI binding sites or inhibition of extracellular signal regulated kinase (ERK) suppressed IRS2 promoter activity almost completely, revealing a major role of MAP kinases (MAPK) for IRS2 transcription. Activating this cascade with oxidative stress increased IRS2 promoter activity and endogenous IRS2 expression substantially. IRS2 promoter activity rose even more after additional inhibition of p38MAPK indicating an inhibitory effect of p38MAPK on ERK mediated IRS2 transcription. Activation of the MAPK pathway using interleukin 1, beta (IL1B) increased IRS2 promoter activity similar to oxidative stress. In contrast IL1B decreases and inhibition of the MAPK pathway increases IRS1 promoter activity revealing opposed effects of IL1B and ERK on the expression of different IRS proteins. In conclusion we discovered a specific region (-688 to -611 bp) in the IRS2 promoter essential for basal promoter activity and oxidative stress induced transcription depending on ERK activation and SP1 and NFI binding in human hepatocytes.
Journal of Molecular Endocrinology 09/2009; 44(2):99-113. · 3.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Alzheimer's disease (AD) is characterized by progressive neurodegeneration leading to loss of cognitive abilities and ultimately to death. Postmortem investigations revealed decreased expression of cerebral insulin-like growth factor (IGF)-1 receptor (IGF-1R) and insulin receptor substrate (IRS) proteins in patients with AD. To elucidate the role of insulin/IGF-1 signaling in AD, we crossed mice expressing the Swedish mutation of amyloid precursor protein (APP(SW), Tg2576 mice) as a model for AD with mice deficient for either IRS-2, neuronal IGF-1R (nIGF-1R(-/-)), or neuronal insulin receptor (nIR(-/-)), and analyzed survival, glucose, and APP metabolism. In the present study, we show that IRS-2 deficiency in Tg2576 mice completely reverses premature mortality in Tg2576 females and delays beta-amyloid (Abeta) accumulation. Analysis of APP metabolism suggested that delayed Abeta accumulation resulted from decreased APP processing. To delineate the upstream signal responsible for IRS-2-mediated disease protection, we analyzed mice with nIGF-1R or nIR deficiency predominantly in the hippocampus. Interestingly, both male and female nIGF-1R(-/-)Tg2576 mice were protected from premature death in the presence of decreased Abeta accumulation specifically in the hippocampus formation. However, neuronal IR deletion had no influence on lethality of Tg2576 mice. Thus, impaired IGF-1/IRS-2 signaling prevents premature death and delays amyloid accumulation in a model of AD.
The FASEB Journal 07/2009; 23(10):3315-24. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Insulin-like growth factor (IGF)-1 increases proliferation, inhibits apoptosis and promotes differentiation of oligodendrocytes and their precursor cells, indicating an important function for IGF-1 receptor (IGF-1R) signaling in myelin development. The insulin receptor substrates (IRS), IRS-1 and -2 serve as intracellular IGF-1R adaptor proteins and are expressed in neurons, oligodendrocytes and their precursors. To address the role of IRS-2 in myelination, we analyzed myelination in IRS-2 deficient (IRS-2(-/-)) mice and age-matched controls during postnatal development. Interestingly, expression of the most abundant myelin proteins, myelin basic protein and proteolipid protein was reduced in IRS-2(-/-) brains at postnatal day 10 (P10) as compared to controls. myelin basic protein immunostaining in P10-IRS-2(-/-) mice revealed a reduced immunostaining, but an unchanged regional distribution pattern. In cerebral myelin isolates at P10 unaltered relative expression of different myelin proteins was found, indicating quantitatively reduced but not qualitatively altered myelination. Interestingly, up-regulation of IRS-1 expression and increased IGF-1R signaling were observed in IRS-2(-/-) mice at P10-14, indicating a compensatory mechanism to overcome IRS-2 deficiency. Adult IRS-2(-/-) mice showed unaltered myelination and motor function. Furthermore, in neuronal/brain-specific insulin receptor knockout mice myelination was unchanged. Thus, our experiments reveal that IGF-1R/IRS-2 mediated signals are critical for appropriate timing of myelination in vivo.
Journal of Neurochemistry 09/2008; 107(4):907-17. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Generation of new adipocytes plays a major role in the development of obesity. We previously have shown that transcriptional repressor factor that binds to IST (FBI)-1 exerts a dual effect in the process of adipogenesis by inhibiting proliferation and promoting differentiation of preadipocytes. The aim of the present study was to identify FBI-1 regulated molecular effectors that could account for these effects. Overexpressing FBI-1 in preadipocytes resulted in reduced expression of the cell cycle regulator cyclin A, which may explain FBI-1 induced inhibition of proliferation. Interestingly, FBI-1 repressed cyclin A promoter activity through an indirect mechanisms that did not involve direct binding of FBI-1 to the promoter sequence, but rather FBI-1 inhibition of transcriptional activator Sp1 binding to a regulatory element at -452 to -443. We also show that FBI-1 promotes terminal preadipocyte differentiation through a mechanism involving decreased levels of expression of the PPARgamma inhibitor E2F-4. FBI-1 significantly reduced E2F-4 promoter activity. Contrary to cyclin A, we found FBI-1-induced repression of E2F-4 is mediated by a direct mechanism via a FBI-1 regulatory element at -11 to -5. As function of transcriptional repressors normally depends on the presence of regulatory co-factors we also performed expression profiling of potential FBI-1 co-repressors throughout adipogenesis. In these experiments Sin3A and histon deacetylase (HDAC)-1 showed a similar expression pattern compared to FBI-1. Strikingly, co-immunoprecipitation studies revealed that FBI-1 binds Sin3A and HDAC-1 to form a repressor complex. Furthermore, by mutational analysis the amino terminal Poxvirus (POZ) domain of FBI-1 was found to be important for Sin3A and HDAC-1 binding. Taken together, FBI-1 is the first transcriptional repressor shown to act as a dual regulator in adipogenesis exerting repressor activities on target genes by both, direct and indirect mechanisms.
Journal of Molecular Medicine 06/2008; 86(5):597-608. · 4.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cerebral insulin receptors play an important role in regulation of energy homeostasis and development of neurodegeneration. Accordingly, type 2 diabetes characterized by insulin resistance is associated with an increased risk of developing Alzheimer's disease. Formation of neurofibrillary tangles, which contain hyperphosphorylated tau, represents a key step in the pathogenesis of neurodegenerative diseases. Here, we directly addressed whether peripheral hyperinsulinemia as one feature of type 2 diabetes can alter in vivo cerebral insulin signaling and tau phosphorylation. Peripheral insulin stimulation rapidly increased insulin receptor tyrosine phosphorylation, mitogen-activated protein kinase and phosphatidylinositol (PI) 3-kinase pathway activation, and dose-dependent tau phosphorylation at Ser202 in the central nervous system. Phospho-FoxO1 and PI-3,4,5-phosphate immunostainings of brains from insulin-stimulated mice showed neuronal staining throughout the brain, not restricted to brain areas without functional blood-brain barrier. Importantly, in insulin-stimulated neuronal/brain-specific insulin receptor knockout mice, cerebral insulin receptor signaling and tau phosphorylation were completely abolished. Thus, peripherally injected insulin directly targets the brain and causes rapid cerebral insulin receptor signal transduction and site-specific tau phosphorylation in vivo, revealing new insights into the linkage of type 2 diabetes and neurodegeneration.