Age-Dependent Changes of Adipokine and Cytokine Secretion From Rat Adipose Tissue by Endogenous and Exogenous Toll-Like Receptor Agonists.

White adipose tissue but recently also brown adipose tissue have emerged as endocrine organs. Age-associated obesity is accompanied by prolonged and elevated lipopolysaccharide (LPS)-induced sickness symptoms and increased cytokine and adipokine levels in the circulation partially originating from adipose tissue. In the present study, ex vivo fat explants were used to investigate how the exogenous pathogen-associated molecular pattern (PAMP) LPS or the endogenous danger-associated molecular patterns (DAMPs) high mobility group box-1 protein (HMGB1) and biglycan modulate the release of cytokines and adipokines/batokines and, thus, could influence systemic and/or local inflammation. The response of adipose tissue (epididymal, retroperitoneal, subcutaneous, and brown) was compared between young lean and old obese rats (2 vs. 24 months old). LPS induced a strong interleukin (IL)-6 and tumor necrosis factor (TNF) alpha release into the supernatant of all adipose tissue types investigated. HMGB1 (subcutaneous) and biglycan (retroperitoneal) led to an increased release of IL-6 and TNFalpha (HMGB1) and decreased visfatin and adiponectin (biglycan) secretion from epididymal adipose tissue (young rats). Visfatin was also decreased by HMGB1 in retroperitoneal adipose tissue of old rats. We found significantly higher leptin (all fat pads) and adiponectin (subcutaneous) levels in supernatants of adipose tissue from old compared to young rats, whereas visfatin secretion showed the opposite. The expression of the biglycan receptor Toll-like receptor (TLR) 2 as well as the LPS and HMGB1 receptors TLR4 and receptor for advanced glycation end products (RAGE) were reduced with age (TLR4/RAGE) and by stimulation with their ligands (subcutaneous). Overall, we revealed that adipokines/adipose-tissue released cytokines show some modulation of their release caused by mediators of septic (batokines) and sterile inflammation with potential implication for acute and chronic disease. Moreover, aging may increase or decrease the release of fat-derived mediators. These data show that DAMPS and LPS locally modulate cytokine secretion while only DAMPS but not LPS can locally alter adipokine secretion during inflammation.

Age Dependent Hypothalamic and Pituitary Responses to Novel Environment Stress or Lipopolysaccharide in Rats.

Previously, we have shown that the transcription factor nuclear factor interleukin (NF-IL)6 can be used as an activation marker for inflammatory lipopolysaccharide (LPS)-induced and psychological novel environment stress (NES) in the rat brain. Here, we aimed to investigate age dependent changes of hypothalamic and pituitary responses to NES (cage switch) or LPS (100 µg/kg) in 2 and 24 months old rats. Animals were sacrificed at specific time points, blood and brains withdrawn and analyzed using immunohistochemistry, RT-PCR and bioassays. In the old rats, telemetric recording revealed that NES-induced hyperthermia was enhanced and prolonged compared to the young group. Plasma IL-6 levels remained unchanged and hypothalamic IL-6 mRNA expression was increased in the old rats. Interestingly, this response was accompanied by a significant upregulation of corticotropin-releasing hormone mRNA expression only in young rats after NES and overall higher plasma corticosterone levels in all aged animals. Immunohistochemical analysis revealed a significant upregulation of NF-IL6-positive cells in the pituitary after NES or LPS-injection. In another important brain structure implicated in immune-to-brain communication, namely, in the median eminence (ME), NF-IL6-immunoreactivity was increased in aged animals, while the young group showed just minor activation after LPS-stimulation. Interestingly, we found a higher amount of NF-IL6-CD68-positive cells in the posterior pituitary of old rats compared to the young counterparts. Moreover, aging affected the regulation of cytokine interaction in the anterior pituitary lobe. LPS-treatment significantly enhanced the secretion of the cytokines IL-6 and TNFα into supernatants of primary cell cultures of the anterior pituitary. Furthermore, in the young rats, incubation with IL-6 and IL-10 antibodies before LPS-stimulation led to a robust decrease of IL-6 production and an increase of TNFα production by the pituitary cells. In the old rats, this specific cytokine interaction could not be detected. Overall, the present results revealed strong differences in the activation patterns and pathways between old and young rats after both stressors. The prolonged hyperthermic and inflammatory response seen in aged animals seems to be linked to dysregulated pituitary cytokine interactions and brain cell activation (NF-IL6) in the hypothalamus-pituitary-adrenal axis.

Leptin is involved in age-dependent changes in response to systemic inflammation in the rat.

Obesity contributes to a state of subclinical peripheral and central inflammation and is often associated with aging. Here we investigated the source and contribution of adipose tissue derived cytokines and the cytokine-like hormone leptin to age-related changes in lipopolysaccharide (LPS)-induced brain-controlled sickness-responses. Old (24 months) and young (2 months) rats were challenged with LPS or saline alone or in combination with a neutralizing leptin antiserum (LAS) or control serum. Changes in the sickness-response were monitored by biotelemetry. Additionally, ex vivo fat-explants from young and old rats were stimulated with LPS or saline and culture medium collected and analyzed by cytokine-specific bioassays/ELISAs. We found enhanced duration/degree of the sickness-symptoms, including delayed but prolonged fever in old rats. This response was accompanied by increased plasma-levels of interleukin (IL)-6 and IL-1ra and exaggerated expression of inflammatory markers in brain and liver analyzed by RT-PCR including inhibitor κBα, microsomal prostaglandin synthase and cyclooxygenase 2 (brain). Moreover, for the first time, we were able to show prolonged elevated plasma leptin-levels in LPS-treated old animals. Treatment with LAS in young rats tended to attenuate the early- and in old rats the prolonged febrile response. Fat-explants exhibited unchanged IL-6 but reduced IL-1ra and tumor necrosis factor (TNF)-α release from adipose tissue of aged compared to young animals. In addition, we found increased expression of the endogenous immune regulator microRNA146a in aged animals suggesting a role for these mediators in counteracting brain inflammation. Overall, our results indicate a role of adipose tissue and leptin in "aging-related-inflammation" and age-dependent modifications of febrile-responses.