Background: Inflammaging represents an accepted concept where the immune system shifts to a low-grade chronic pro-inflammatory state without overt infection upon aging. In the CNS, inflammaging is mainly driven by glia cells and associated with neurodegenerative processes. White matter degeneration (WMD), a well-known process in the aging brain, manifests in myelin loss finally resulting in motor, sensory and cognitive impairments. Oligodendrocytes (OL) are responsible for homeostasis and maintenance of the myelin sheaths, which is a complex and highly energy demanding process sensitizing these cells to metabolic, oxidative and other forms of stress. Yet, the immediate impact of chronic inflammatory stress like inflammaging on OL homeostasis, myelin maintenance and WMD remains open. Methods: To functionally analyze the role of IKK/NF-κB signaling in the regulation of myelin homeostasis and maintenance in the adult CNS, we established a conditional mouse model allowing NF-κB activation in mature myelinating oligodendrocytes. IKK2-CA^{PLP−CreERT2} mice were characterized by biochemical, immunohistochemical, ultrastructural and behavioral analyses. Transcriptome data from isolated, primary OLs and microglia cells were explored by in silico pathway analysis and validated by complementary molecular approaches. Results: Chronic NF-κB activation in mature OLs leads to aggravated neuroinflammatory conditions phenocopying brain inflammaging. As a consequence, IKK2-CA^{PLP−CreERT2} mice showed specific neurological deficits and impaired motoric learning. Upon aging, persistent NF-κB signaling promotes WMD in these mice as ultrastructural analysis revealed myelination deficits in the corpus callosum accompanied by impaired myelin protein expression. RNA-Seq analysis of primary oligodendrocytes and microglia cells uncovers gene expression signatures associated with activated stress responses and increased post mitotic cellular senescence (PoMiCS) which was confirmed by elevated senescence-associated β-galactosidase activity and SASP gene expression profile. We identified an elevated integrated stress response (ISR) characterized by phosphorylation of eIF2α as a relevant molecular mechanism which is able to affect translation of myelin proteins. Conclusions: Our findings demonstrate an essential role of IKK/NF-κB signaling in mature, post-mitotic OLs in regulating stress-induced senescence in these cells. Moreover, our study identifies PoMICS as an important driving force of age-dependent WMD as well as of traumatic brain injury induced myelin defects. Graphical

Brown adipose tissue (BAT) has been considered beneficial for metabolic health by participating in the regulation of glucose homoeostasis. The browning factors that improve glucose uptake beyond normal levels are still unknown but glucose uptake is not affected in UCP1 knockout mice. Here, we demonstrate in human white adipocytes that basal/resting glucose uptake is improved by solely elevating UCP1 protein levels. Generating human white Simpson-Golabi-Behmel syndrome (SGBS) adipocytes with a stable knockout and overexpression of UCP1, we discovered that UCP1 overexpressing adipocytes significantly improve glucose uptake by 40%. Mechanistically, this is caused by higher glycolytic flux, seen as increased oxygen consumption, extracellular acidification and lactate secretion rates. The improvements in glucose handling are comparable to white-to-brown transitions, as judged by, for the first time, directly comparing in vitro differentiated mouse brown vs white adipocytes. Although no adipogenic, metabolic and mitochondrial gene expressions were significantly altered in SGBS cells, pharmacological inhibition of GLUT1 completely abrogated differences between UCP1+ and control cells, thereby uncovering GLUT1-mediated uptake as permissive gatekeeper. Collectively, our data demonstrate that elevating UCP1 levels is sufficient to improve human white adipocytes as a glucose sink without adverse cellular effects, thus not requiring the adrenergic controlled, complex network of browning which usually hampers translational efforts.

MicroRNAs (miRNAs), a class of small, non-coding RNA molecules, play an important role in the posttranscriptional regulation of gene expression, thereby influencing important cellular functions. In adipocytes, miRNAs show import regulatory features and are described to influence differentiation as well as metabolic, endocrine, and inflammatory functions. We previously identified miR-27a being upregulated under inflammatory conditions in human adipocytes and aimed to elucidate its function in adipocyte biology. Both strands of miR-27a, miR-27a-3p and -5p, were downregulated during the adipogenic differentiation of Simpson–Golabi–Behmel syndrome (SGBS) cells, human multipotent adipose-derived stem cells (hMADS), and human primary adipose-derived stromal cells (hASCs). Using miRNA-mimic transfection, we observed that miR-27a-3p is a crucial regulator of adipogenesis, while miR-27a-5p did not alter the differentiation capacity in SGBS cells. In silico screening predicted lipoprotein lipase (LPL) and peroxisome proliferator activated receptor γ (PPARγ) as potential targets of miR-27a-3p. The downregulation of both genes was verified in vitro, and the interaction of miR-27-3p with target sites in the 3′ UTRs of both genes was confirmed via a miRNA-reporter-gene assay. Here, the knockdown of LPL did not interfere with adipogenic differentiation, while PPARγ knockdown decreased adipogenesis significantly, suggesting that miR-27-3p exerts its inhibitory effect on adipogenesis by repressing PPARγ. Taken together, we identified and validated a crucial role for miR-27a-3p in human adipogenesis played by targeting the essential adipogenic transcription factor PPARγ. Though we confirmed LPL as an additional target of miR-27a-3p, it does not appear to be involved in regulating human adipogenesis. Thereby, our findings call the conclusions drawn from previous studies, which identified LPL as a crucial regulator for murine and human adipogenesis, into question.

Functional brown adipose tissue (BAT), involved in energy expenditure, has recently been detected in substantial amounts in adults. Formerly overlooked BAT has now become an attractive anti-obesity target. Molecular characterization of human brown and white adipocytes, using a myriad of techniques including high-throughput RNA sequencing and functional assays, showed that PAZ6 and SW872 cells exhibit classical molecular and phenotypic markers of brown and white adipocytes, respectively. However, the pre-adipocyte cell line SGBS presents a versatile phenotype. A transit expression of classical brown markers such as UCP1 and PPARγ peaked and declined at day 28 post-differentiation initiation. Conversely, white adipocyte markers, including Tcf21, showed reciprocal behavior. Interestingly, leptin levels peaked at day 28 whereas the highest adiponectin mRNA levels were detected at day 14 of differentiation. Phenotypic analysis of the abundance and shape of lipid droplets were consistent with the molecular patterns. Accordingly, the oxidative capacity of SGBS adipocytes peaked on differentiation day 14 and declined progressively towards differentiation day 28. Our studies have unveiled a new phenotype of human adipocytes, providing a tool to identify molecular gene expression patterns and pathways involved in the conversion between white and brown adipocytes.

Obesity is a persistent and continuously expanding social health concern. Excessive fat mass accumulation is associated with increased risk of chronic diseases including diabetes, atherosclerosis, non-alcoholic steatohepatitis, reproductive dysfunctions and certain types of cancer. Alchemilla monticola Opiz. is a perennial plant of the Rosaceae family traditionally used to treat inflammatory conditions and as a component of weight loss herbal mixtures. In the search for bioactive leads with potential anti-adipogenic effect from A. monticola extract (ALM), we have employed nuclear magnetic resonance (NMR) based metabolomics to obtain data for the phytochemical profile of the extract. Further, molecular docking simulation was performed against key adipogenic targets for selected pure compounds, present in the ALM extract. Evaluation of the biological activity was done in human adipocytes exposed to ALM (5, 10 and 25 μg/ml), pure astragalin (AST) or quercitrin (QUE) both at the concentrations of 5, 10 and 25 μM. Investigation of the molecular pathways involved was performed through real-time quantitative PCR and Western blot analyses. According to the docking predictions strong putative affinity was revealed for both AST and QUE towards peroxisome proliferator-activated receptor gamma (PPARγ) and phosphoinositide 3-kinase (PI3K). Assessment of the intracellular lipid accumulation revealed anti-adipogenic activity of ALM. Correspondingly, the expression of the adipogenic genes CCAAT/enhancer-binding protein alpha (CEBPA) and PPARG was downregulated upon ALM and AST treatment. The Western blotting results exposed protein kinase B (AKT), PI3K and PPARγ as targets for the inhibitory effect of ALM and AST on adipogenesis. Collectively, we provide a broader insight of the phytochemical composition of A. monticola. Additionally, we demonstrate the anti-adipogenic effect of ALM and its active compound AST in human adipocytes. Furthermore, PI3K/AKT signaling pathway is identified to mediate the ALM anti-adipogenic action. Hence, the ALM extract and its secondary metabolite AST are worth further exploration as potentially active agents in obesity management.