Research reportInsulin activates microglia and increases COX-2/IL-1β expression in young but not in aged hippocampus
Graphical abstract
Introduction
Microglia are a specialized and dynamic population of central nervous system (CNS) parenchyma-resident macrophages that are able to detect tissue damage and (micro) environmental variations (Hanisch and Kettenmann, 2007). Although, microglia are the primary innate immune cells in the brain, their CNS-tailored functions go beyond their well-characterized roles in neuroinflammation (Salter and Beggs, 2014). Microglia also have important functions in non-immune brain physiology and are reported to contribute to learning and memory (Parkhurst et al., 2013). Other important microglia functions are trophic neuronal support, removal of cellular debris, synaptic pruning, injury repair and homeostatic support for living neurons, which is crucial for the neural circuit remodeling and synaptic plasticity (Galatro et al., 2017, Janssen et al., 2017, Salter and Beggs, 2014).
Microglia sense disturbances in brain homeostasis and turn out to be “activated”, a phenotype characterized by morphological changes, e.g. process retraction and thickening, and upregulation of a broad spectrum of cellular membrane surface antigens, such as CD68 (O'Neil et al., 2018). Microglial activation is accompanied by expression of several intracellular molecules including the COX enzyme family followed by intracellular and extracellular expression of cytokines (Akundi et al., 2005). Neuroprotective functions of COX-2 are already described, which include contributions to learning and memory processing (Bazan, 2006, Choi et al., 2009, Teather et al., 2002). Although substantial amount of evidences had identified both IL-1β and IL-6 as important cytokines with potential neuroprotective functions (Borsini et al., 2015, Frakes et al., 2014, Gruol, 2015, Seibert and Masferrer, 1994, Tak and Firestein, 2001, Takemiya et al., 2017) indicating that proinflammatory cytokines responses are not only detrimental to the brain (Biber et al., 2014), IL-1β and IL-6 neurophysiological and neuroprotective signals still need further scrutiny.
During aging, microglia adopt a more activated and sensitized state phenotype referred to a “primed”, resulting in an exaggerated inflammatory response to an inflammatory stimulus (Perry and Holmes, 2014, Raj et al., 2014). Aging is associated with persistent changes in the brain microenvironment, and chronic neuroinflammatory stimulation has been proposed as a key factor associated with microglial dysfunction and a mechanism for inducing the age-related functional deficits (Holtman et al., 2015, Karch and Goate, 2015, Norden et al., 2015, Spittau, 2017). However, although aged microglia may adopt an undesirable phenotype, the mechanistic effectors of this reprogramming are not well comprehended.
Insulin has diverse functions in the CNS, including neurotrophic signaling (Bomfim et al., 2012, Rhea et al., 2019). Brain insulin administration improved memory in young but not in aged rats, which was related with a disrupted connection between both insulin and BDNF pathways in the aged hippocampus (Haas et al., 2016). Accordingly, experimental rodent models and studies with aged patients have consistently demonstrated a strong mechanistic association between deficient insulin signaling, cognitive disruption and Alzheimer’s disease (Ghasemi et al., 2013, Gutchess, 2014, Lopez-Otin et al., 2013, Steculorum et al., 2014, Talbot et al., 2012). The role of insulin in neurons has been extensively investigated, but only recently its role in glial cells has received the necessary attention. For instance, insulin signaling in hypothalamic astrocytes controls the availability of glucose to neurons, which is crucial for the regulation of whole body energy homeostasis (Garcia-Caceres et al., 2016). Recently, it was demonstrated that insulin receptor (IR) and upstream proteins (IRS-1 and IRS-2) are expressed in human primary astrocytes and microglia supporting the notion that insulin may directly bind to and influence microglia activity (Spielman et al., 2015), but, it is still unclear the downstream microglial insulin signaling (Akt-GSK3-β), how insulin affects microglia activation in vivo, and, similarly as occurs in aged neurons (Rodriguez-Rodriguez et al., 2017), if primed microglia are resistant to insulin.
In order to address the effect of insulin on microglia in vivo, we performed insulin infusions directly into the brain of young and aged rats and analyzed their activation, and COX-2, IL-6 and IL-1β expression in the hippocampus. To detect a direct insulin-mediated response by microglia, we performed in vitro experiments using primary microglia cultures.
Section snippets
Insulin triggers microglia activation in the young but not in the aged hippocampus
To evaluate the insulin effects on microglia in vivo, we performed Iba-1/CD68 co-staining (Fig. 1a) to access microglia activation in the hippocampus. We found that after five days of i.c.v. insulin treatment, microglia of young rats were more activated in CA1 (p = 0.022) (Fig. 1b) and CA3 (p = 0.009) (Fig. 1c) hippocampal subregions when compared to controls. No significant alterations were detected in the DG (p = 0.165) (Fig. 1d). During aging, CNS develops resistance to insulin signaling and
Discussion
Our findings imply that the inflammatory responses to insulin are different between young and aged microglia. This notion is supported by the increased number of CD68-positive microglia in the CA1 and CA3 hippocampal subfields of young but not in aged rats in response to insulin. Complementary, the analysis of the inflammatory markers associated with microglia highlighted a concomitant higher expression of COX-2 and IL-1β in the hippocampus of young rats, and these elevated COX-2 levels were
In vivo experiments
In vivo experiments were performed using 3 and 22-month-old male Wistar rats under approval of the UFRGS Local Ethic Committee for Animal Experimentation (CEUA); project number: #26376. Wistar rats were housed in groups of maximum five animals, in a room with controlled temperature, under a 12 h light/dark cycle and with free access to food and water.
Acknowledgements
This work was supported by the Brazilian grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq #426796/2016-0 and #141100/2013-3, CNPq – Instituto Nacional de Neurociência Translacional – INNT #465346/2014-6, Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul – FAPERGS – PRONEX #16/2551-0000499-4. This study was also funded by the Abel Tasman Talent Program – Graduate School of Medical Sciences sponsorship and Jan Kornelis de Cock Stichting grant awarded
Conflict of interest
The authors state that no personal, scientific or financial conflict of interest exists.
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