Kirsten P. Stone

Cytokine signaling modulates blood-brain barrier function.

The blood-brain barrier (BBB) provides a vast interface for cytokines to affect CNS function. The BBB is a target for therapeutic intervention. It is essential, therefore, to understand how cytokines interact with each other at the level of the BBB and how secondary signals modulate CNS functions beyond the BBB. The interactions between cytokines and lipids, however, have not been fully addressed at the level of the BBB. Here, we summarize current understanding of the localization of cytokine receptors and transporters in specific membrane microdomains, particularly lipid rafts, on the luminal (apical) surface of the microvascular endothelial cells composing the BBB. We then illustrate the clinical context of cytokine effects on the BBB by neuroendocrine regulation and amplification of inflammatory signals. Two unusual aspects discussed are signaling crosstalk by different classes of cytokines and genetic regulation of drug efflux transporters. We also introduce a novel area of focus on how cytokines may act through nuclear hormone receptors to modulate efflux transporters and other targets. A specific example discussed is the ATP-binding cassette transporter-1 (ABCA-1) that regulates lipid metabolism. Overall, cytokine signaling at the level of the BBB is a crucial feature of the dynamic regulation that can rapidly change BBB function and affect brain health and disease.

Mechanisms of Increased In Vivo Insulin Sensitivity by Dietary Methionine Restriction in Mice

To understand the physiological significance of the reduction in fasting insulin produced by dietary methionine restriction (MR), hyperinsulinemic-euglycemic clamps were used to examine the effect of the diet on overall and tissue-specific insulin sensitivity in mice. The steady-state glucose infusion rate was threefold higher in the MR group and consistent with the 2.5- to threefold increase in 2-deoxyglucose uptake in skeletal muscle, heart, and white adipose tissue. Dietary MR enhanced suppression of hepatic glucose production by insulin, enhanced insulin-dependent Akt phosphorylation in the liver, and increased hepatic expression and circulating fibroblast growth factor 21 (FGF-21) by fourfold. Limitation of media methionine recapitulated amplification of Akt phosphorylation by insulin in HepG2 cells but not in 3T3-L1 adipocytes or C2C12 myotubes. Amplification of insulin signaling in HepG2 cells by MR was associated with reduced glutathione, where it functions as a cofactor for phosphatase and tensin homolog. In contrast, FGF-21, but not restricting media methionine, enhanced insulin-dependent Akt phosphorylation in 3T3-L1 adipocytes. These findings provide a potential mechanism for the diet-induced increase in insulin sensitivity among tissues that involves a direct effect of methionine in liver and an indirect effect in adipose tissue through MR-dependent increases in hepatic transcription and release of FGF-21.

Remodeling the Integration of Lipid Metabolism Between Liver and Adipose Tissue by Dietary Methionine Restriction in Rats

Dietary methionine restriction (MR) produces an integrated series of biochemical and physiological responses that improve biomarkers of metabolic health, limit fat accretion, and enhance insulin sensitivity. Using transcriptional profiling to guide tissue-specific evaluations of molecular responses to MR, we report that liver and adipose tissue are the primary targets of a transcriptional program that remodeled lipid metabolism in each tissue. The MR diet produced a coordinated downregulation of lipogenic genes in the liver, resulting in a corresponding reduction in the capacity of the liver to synthesize and export lipid. In contrast, the transcriptional response in white adipose tissue (WAT) involved a depot-specific induction of lipogenic and oxidative genes and a commensurate increase in capacity to synthesize and oxidize fatty acids. These responses were accompanied by a significant change in adipocyte morphology, with the MR diet reducing cell size and increasing mitochondrial density across all depots. The coordinated transcriptional remodeling of lipid metabolism between liver and WAT by dietary MR produced an overall reduction in circulating and tissue lipids and provides a potential mechanism for the increase in metabolic flexibility and enhanced insulin sensitivity produced by the diet.

NFĸB is an unexpected major mediator of interleukin-15 signaling in cerebral endothelia.

Interleukin (IL)-15 and its receptors are induced by tumor necrosis factor α (TNF) in the cerebral endothelial cells composing the blood-brain barrier, but it is not yet clear how IL-15 modulates endothelial function. Contrary to the known induction of JAK/STAT3 signaling, here we found that nuclear factor (NF)- ĸB is mainly responsible for IL-15 actions on primary brain microvessel endothelial cells and cerebral endothelial cell lines. IL-15-induced transactivation of an NFĸB luciferase reporter resulted in phosphorylation and degradation of the inhibitory subunit IĸB that was followed by phosphorylation and nuclear translocation of the p65 subunit of NFĸB. An IĸB kinase inhibitor Bay 11-7082 only partially inhibited IL-15-induced NFĸB luciferase activity. The effect of IL-15 was mediated by its specific receptor IL-15Rα, since endothelia from IL-15Rα knockout mice showed delayed nuclear translocation of p65, whereas those from knockout mice lacking a co-receptor IL-2Rγ did not show such changes. At the mRNA level, IL-15 and TNF showed similar effects in decreasing the tight junction protein claudin-2 and increasing the p65 subunit of NFĸB but exerted different regulation on caveolin-1 and vimentin. Taken together, NFĸB is a major signal transducer by which IL-15 affects cellular permeability, endocytosis, and intracellular trafficking at the level of the blood-brain barrier.

UCP1 is an essential mediator of the effects of methionine restriction on energy balance but not insulin sensitivity

Dietary methionine restriction (MR) by 80% increases energy expenditure (EE), reduces adiposity, and improves insulin sensitivity. We propose that the MR‐induced increase in EE limits fat deposition by increasing sympathetic nervous system‐dependent remodeling of white adipose tissue and increasing uncoupling protein 1 (UCP1) expression in both white and brown adipose tissue. In independent assessments of the role of UCP1 as a mediator of MRs effects on EE and insulin sensitivity, EE did not differ between wild‐type (WT) and Ucp1‐/‐ mice on the control diet, but MR increased EE by 31 % and reduced adiposity by 25% in WT mice. In contrast, MR failed to increase EE or reduce adiposity in Ucp1‐/‐ mice. However, MR was able to increase overall insulin sensitivity by 2.2‐fold in both genotypes. Housing temperatures used to minimize (28°C) or increase (23°C) sympathetic nervous system activity revealed temperature‐independent effects of the diet on EE. Metabolomics analysis showed that genotypic and dietary effects on white adipose tissue remodeling resulted in profound increases in fatty acid metabolism within this tissue. These findings establish that UCP1 is required for the MR‐induced increase in EE but not insulin sensitivity and suggest that diet‐induced improvements in insulin sensitivity are not strictly derived from dietary effects on energy balance.—Wanders, D., Burk, D. H., Cortez, C. C., Van, N. T., Stone, K. P., Baker, M., Mendoza, T., Mynatt, R. L., Gettys, T. W. UCP1 is an essential mediator of the effects of methionine restriction on energy balance but not insulin sensitivity. FASEB J. 29, 2603‐2615 (2015). www.fasebj.org

The Impact of Dietary Methionine Restriction on Biomarkers of Metabolic Health

Calorie restriction without malnutrition, commonly referred to as dietary restriction (DR), results in a well-documented extension of life span. DR also produces significant, long-lasting improvements in biomarkers of metabolic health that begin to accrue soon after its introduction. The improvements are attributable in part to the effects of DR on energy balance, which limit fat accumulation through reduction in energy intake. Accumulation of excess body fat occurs when energy intake chronically exceeds the energy costs for growth and maintenance of existing tissue. The resulting obesity promotes the development of insulin resistance, disordered lipid metabolism, and increased expression of inflammatory markers in peripheral tissues. The link between the life-extending effects of DR and adiposity is the subject of an ongoing debate, but it is clear that decreased fat accumulation improves insulin sensitivity and produces beneficial effects on overall metabolic health. Over the last 20 years, dietary methionine restriction (MR) has emerged as a promising DR mimetic because it produces a comparable extension in life span, but surprisingly, does not require food restriction. Dietary MR also reduces adiposity but does so through a paradoxical increase in both energy intake and expenditure. The increase in energy expenditure fully compensates for increased energy intake and effectively limits fat deposition. Perhaps more importantly, the diet increases metabolic flexibility and overall insulin sensitivity and improves lipid metabolism while decreasing systemic inflammation. In this chapter, we describe recent advances in our understanding of the mechanisms and effects of dietary MR and discuss the remaining obstacles to implementing MR as a treatment for metabolic disease.

Role of GCN2-Independent Signaling Through a Noncanonical PERK/NRF2 Pathway in the Physiological Responses to Dietary Methionine Restriction.

Restricting availability of essential amino acids (EAAs) limits aminoacylation of tRNAs by their cognate EAAs and activates the nutrient-sensing kinase, general control nonderepressible 2 (GCN2). Activated GCN2 phosphorylates eukaryotic initiation factor 2 (eIF2), altering gene-specific translation and initiating a transcriptional program collectively described as the integrated stress response (ISR). Central GCN2 activation by EAA deprivation is also linked to an acute aversive feeding response. Dietary methionine restriction (MR) produces a well-documented series of physiological responses (increased energy intake and expenditure, decreased adiposity, and increased insulin sensitivity), but the role of GCN2 in mediating them is unknown. Using Gcn2−/− mice, we found that the absence of GCN2 had no effect on the ability of MR to reduce body weight or adiposity, increase energy intake and expenditure, increase hepatic transcription and release of fibroblast growth factor 21, or improve insulin sensitivity. Interestingly, hepatic eIF2 phosphorylation by MR was uncompromised in Gcn2−/− mice. Instead, protein kinase R–like endoplasmic reticulum (ER) kinase (PERK) was activated in both intact and Gcn2−/− mice. PERK activation corresponded with induction of the ISR and the nuclear respiratory factor 2 antioxidant program but not ER stress. These data uncover a novel glutathione-sensing mechanism that functions independently of GCN2 to link dietary MR to its metabolic phenotype.

Transcriptional impact of dietary methionine restriction on systemic inflammation: relevance to biomarkers of metabolic disease during aging.

Calorie restriction (CR) without malnutrition increases lifespan and produces significant improvements in biomarkers of metabolic health. The improvements are attributable in part to effects of CR on energy balance, which limit fat accumulation by restricting energy intake. Normal age‐associated increases in adiposity and insulin resistance are associated with development of a systemic proinflammatory state, while chronic CR limits fat deposition and expression of inflammatory markers. Dietary methionine restriction (MR) has emerged as an effective CR mimetic because it produces a comparable extension in lifespan. MR also reduces adiposity through a compensatory increase in energy expenditure that effectively limits fat accumulation, but essentially nothing is known about the effects of MR on systemic inflammation. Here, we review the relationships between these two interventions and discuss their transcriptional impact. In addition, using tissues from rats after long‐term consumption of CR or MR diets, transcriptional profiling was used to examine retrospectively the systems biology of 59 networks of molecules annotated to inflammation. Transcriptional effects of both diets occurred primarily in white adipose tissue and liver, and the responses to MR were far more robust than those to CR. The primary transcriptional targets of MR in both liver and white adipose tissue were phagocytes and macrophages, where expression of genes associated with immune cell infiltration and quantity was reduced. These findings support the conclusion that anti‐inflammatory responses produced by CR and MR are not strictly dependent upon reduced adiposity but are significantly influenced by the metabolic mechanisms through which energy balance is altered.

A systems biology analysis of the unique and overlapping transcriptional responses to caloric restriction and dietary methionine restriction in rats

Dietary methionine restriction (MR) and calorie restriction (CR) each improve metabolic health and extend life span. We used comprehensive transcriptome profiling and systems biology analysis to interrogate the unique and overlapping molecular responses in rats provided these dietary regimens for 20 mo after weaning. Microarray analysis was conducted on inguinal white adipose (IWAT), brown adipose tissue (BAT), liver, and skeletal muscle. Compared to controls, CR‐induced transcriptomic responses (absolute fold change ≥1.5 and P≤0.05) were comparable in IWAT, BAT, and liver (~800 genes). MR‐induced effects were largely restricted to IWAT and liver (~2400 genes). Pathway enrichment and gene‐coexpression analyses showed that induction of fatty acid synthesis in IWAT was common to CR and MR, whereas immunity and proinflammatory signaling pathways were specifically down‐regulated in MR‐treated IWAT and liver (FDR≤0.07–0.3). BAT demonstrated consistent down‐regulation of PPAR‐signaling under CR and MR, whereas muscle was largely unaffected. Interactome analysis identified CR‐specific down‐regulation of cytoskeletal matrix components in IWAT and MR‐specific up‐regulation of ribosomal genes in liver (FDR≤0.001). Transcriptomic down‐regulation of inflammation genes by MR in IWAT was consistent with upstream inhibition of STAT3. Together, these results provide an integrated picture of the breadth of transcriptional responses to MR and CR among key metabolic tissues.—Ghosh, S., Wanders, D., Stone, K. P., Van, N. T., Cortez, C. C., Gettys, T. W. A systems biology analysis of the unique and overlapping transcriptional responses to caloric restriction and dietary methionine restriction in rats. FASEB J. 28, 2577–2590 (2014). www.fasebj.org

Interleukin-15 affects serotonin system and exerts antidepressive effects through IL15Rα receptor

Contrary to the reduction of depressive-like behavior observed in several strains of cytokine receptor knockout mice, mice lacking the specific receptor for interleukin (IL)-15 showed increased immobility in tail suspension and modified forced swimming tests. There was also a reduction in social interactions. The hippocampus of the IL15Rα knockout mice had decreased mRNA for 5-HT(1A), increased mRNA for 5-HT(2C), and region-specific changes of serotonin reuptake transporter (SERT) immunoreactivity. Fluoxetine (the classic antidepressant Prozac, which inhibits 5-HT(2C) and SERT) reduced the immobility of the IL15Rα knockout mice in comparison with their pretreatment baseline. Together with the unchanged performance of the IL15Rα knockout mice on the rotarod, this response to fluoxetine indicates that the immobility reflects depression. Wildtype mice responded to IL15 treatment with improvement of immobility induced by forced swimming, whereas the knockout mice failed to respond. Thus, the cognate IL15 receptor is necessary for the antidepressive activity of IL15. In ex vivo studies, IL15 decreased synaptosomal uptake of 5-HT, and modulated the expression of 5-HT(2C) and SERT in cultured neurons in a dose- and time-dependent manner. Thus, the effect of IL15 on serotonin transmission may underlie the depressive-like behavior of IL15Rα knockout mice. We speculate that IL15 is essential to maintain neurochemical homeostasis and thereby plays a role in preventing neuropsychiatric symptoms.