Fructose Alters Cell Survival and Gene Expression in Microglia and Neuronal Cells Lines

Purpose: Microglia are macrophages that are found primarily in the CNS and play a crucial role in maintaining a healthy brain by engulfing invading microorganisms, releasing inflammatory mediators, and pruning dead cells. Microglia can become activated in response to certain stimuli which causes them to transition into a pro-inflammatory state, and can sometimes become chronically activated which can result in neuronal damage. Studies have shown a causal relationship between this activation and sugars such as fructose and glucose. We sought to understand the role of sugars in microglial activation and the subsequent effects on neuron health. Methods: Rat microglia (HAPI) and neuronal (B35) cell lines were treated with varying concentrations of fructose (25 mM, 12.5 mM, and 6.25 mM) or glucose (25 mM and 12.5 mM)as a positive control to determine their effects on the cells. Following treatment and incubation for 3 or 24 hours, the cells were analyzed using an MTT assay to measure cell survival or real-time polymerase chain reaction (RT-PCR) to measure gene expression levels. Effects of fructose were measured in HAPI microglia after direct treatment with the sugar. The genes investigated by the RT-PCR in the HAPI cells included: glucose transporter 5 (GLUT5), and the inflammatory markers high mobility group box 1 (HMGB1), and prostaglandin E receptor 2 (Ptger2). To evaluate the effects of microglial activation on neuronal function, the B35 neurons were treated either directly with sugars or with the supernatant collected from fructose-treated HAPI microglia. This allows examination of the effects of soluble neuron-injury factors released by microglia. The genes investigated by RT-PCR in B35 neurons included nuclear factor-κB (NFκB) and enolase 2 (Eno2). Results: Cell survival assays showed that 24-hour direct fructose treatment increased B35 cell survival by up to 13%, while groups treated with microglia supernatant increased cell survival by up to 33%. In HAPI microglia, 3 hours of treatment with fructose caused GLUT5 expression to be suppressed by up to 32% in all treatment groups except for 6.25 mM fructose, while Ptger2 and HMGB1 expression was increased by as much as 65% and 15%, respectively. After 24-hours of treatment with fructose, the HAPI microglia showed a maximum of 80% increased expression of HMGB1, while Ptger2 expression was mostly unchanged. In B35 neurons, 3 hours of treatment with fructose caused a decrease of up to 26% in NFκB and an increase of up to 46% in Eno2 expression. Conclusion: Cell survival results indicate that the microglia may provide a short term protective effect on the B35 neurons. However, data from the gene expression assays show evidence of cellular dysfunction in neurons and pro-inflammatory activity in microglia which may lead to neuronal death on a longer timeline. As seen in the gene expression results, microglia had increased expression of pro-inflammatory genes and B35 neuronal cells had increased expression of markers of cellular damage. Future studies will further explore the effects of fructose on expression of other genes and examine the effects on neuron survival at later time points

Nox4 Mediates Renal Cell Carcinoma Cell Invasion through Hypoxia-Induced Interleukin 6- and 8- Production

Inflammatory cytokines are detected in the plasma of patients with renal cell carcinoma (RCC) and are associated with poor prognosis. However, the primary cell type involved in producing inflammatory cytokines and the biological significance in RCC remain unknown. Inflammation is associated with oxidative stress, upregulation of hypoxia inducible factor 1-alpha, and production of pro-inflammatory gene products. Solid tumors are often heterogeneous in oxygen tension together suggesting that hypoxia may play a role in inflammatory processes in RCC. Epithelial cells have been implicated in cytokine release, although the stimuli to release and molecular mechanisms by which they are released remain unclear. AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status and a role for AMPK in the regulation of cell inflammatory processes has recently been demonstrated.We have identified for the first time that interleukin-6 and interleukin-8 (IL-6 and IL-8) are secreted solely from RCC cells exposed to hypoxia. Furthermore, we demonstrate that the NADPH oxidase isoform, Nox4, play a key role in hypoxia-induced IL-6 and IL-8 production in RCC. Finally, we have characterized that enhanced levels of IL-6 and IL-8 result in RCC cell invasion and that activation of AMPK reduces Nox4 expression, IL-6 and IL-8 production, and RCC cell invasion.Together, our data identify novel mechanisms by which AMPK and Nox4 may be linked to inflammation-induced RCC metastasis and that pharmacological activation of AMPK and/or antioxidants targeting Nox4 may represent a relevant therapeutic intervention to reduce IL-6- and IL-8-induced inflammation and cell invasion in RCC

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV

Fructose Alters Cell Survival and Gene Expression in Microglia and Neuronal Cells Lines

Purpose: Microglia are macrophages that are found primarily in the CNS and play a crucial role in maintaining a healthy brain by engulfing invading microorganisms, releasing inflammatory mediators, and pruning dead cells. Microglia can become activated in response to certain stimuli which causes them to transition into a pro-inflammatory state, and can sometimes become chronically activated which can result in neuronal damage. Studies have shown a causal relationship between this activation and sugars such as fructose and glucose. We sought to understand the role of sugars in microglial activation and the subsequent effects on neuron health. Methods: Rat microglia (HAPI) and neuronal (B35) cell lines were treated with varying concentrations of fructose (25 mM, 12.5 mM, and 6.25 mM) or glucose (25 mM and 12.5 mM)as a positive control to determine their effects on the cells. Following treatment and incubation for 3 or 24 hours, the cells were analyzed using an MTT assay to measure cell survival or real-time polymerase chain reaction (RT-PCR) to measure gene expression levels. Effects of fructose were measured in HAPI microglia after direct treatment with the sugar. The genes investigated by the RT-PCR in the HAPI cells included: glucose transporter 5 (GLUT5), and the inflammatory markers high mobility group box 1 (HMGB1), and prostaglandin E receptor 2 (Ptger2). To evaluate the effects of microglial activation on neuronal function, the B35 neurons were treated either directly with sugars or with the supernatant collected from fructose-treated HAPI microglia. This allows examination of the effects of soluble neuron-injury factors released by microglia. The genes investigated by RT-PCR in B35 neurons included nuclear factor-κB (NFκB) and enolase 2 (Eno2). Results: Cell survival assays showed that 24-hour direct fructose treatment increased B35 cell survival by up to 13%, while groups treated with microglia supernatant increased cell survival by up to 33%. In HAPI microglia, 3 hours of treatment with fructose caused GLUT5 expression to be suppressed by up to 32% in all treatment groups except for 6.25 mM fructose, while Ptger2 and HMGB1 expression was increased by as much as 65% and 15%, respectively. After 24-hours of treatment with fructose, the HAPI microglia showed a maximum of 80% increased expression of HMGB1, while Ptger2 expression was mostly unchanged. In B35 neurons, 3 hours of treatment with fructose caused a decrease of up to 26% in NFκB and an increase of up to 46% in Eno2 expression. Conclusion: Cell survival results indicate that the microglia may provide a short term protective effect on the B35 neurons. However, data from the gene expression assays show evidence of cellular dysfunction in neurons and pro-inflammatory activity in microglia which may lead to neuronal death on a longer timeline. As seen in the gene expression results, microglia had increased expression of pro-inflammatory genes and B35 neuronal cells had increased expression of markers of cellular damage. Future studies will further explore the effects of fructose on expression of other genes and examine the effects on neuron survival at later time points

LINE- and Alu-containing genomic instability hotspot at 16q24.1 associated with recurrent and nonrecurrent CNV deletions causative for ACDMPV

Transposable elements modify human genome by inserting into new loci or by mediating homology-, microhomology-, or homeology-driven DNA recombination or repair, resulting in genomic structural variation. Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal neonatal developmental lung disorder caused by point mutations or copy-number variant (CNV) deletions of FOXF1 or its distant tissue-specific enhancer. Eighty-five percent of 45 ACDMPV-causative CNV deletions, of which junctions have been sequenced, had at least one of their two breakpoints located in a retrotransposon, with more than half of them being Alu elements. We describe a novel similar to 35 kb-large genomic instability hotspot at 16q24.1, involving two evolutionarily young LINE-1 (L1) elements, L1PA2 and L1PA3, flanking AluY, two AluSx, AluSx1, and AluJr elements. The occurrence of L1s at this location coincided with the branching out of the Homo-Pan-Gorilla clade, and was preceded by the insertion of AluSx, AluSx1, and AluJr. Our data show that, in addition to mediating recurrent CNVs, L1 and Alu retrotransposons can predispose the human genome to formation of variably sized CNVs, both of clinical and evolutionary relevance. Nonetheless, epigenetic or other genomic features of this locus might also contribute to its increased instability