Obesity often leads to increased systemic inflammation which is now thought to play a causative role in the development of atherosclerotic disease and insulin resistance ^{1– 4} . Increasing adiposity due to excessive weight gain sets up a chronic inflammatory response within adipose tissue which is promoted by recruitment and infiltration of classically activated, type-1 macrophages from the circulation ⁵ . Although the reasons for sustained adipose inflammation remain unclear, the large number of macrophages residing in obese adipose tissue leads to significant increases in secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα); achieving levels sufficient to elevate circulating plasma concentrations ⁶ . IL-6 ⁷ and TNFα ^{8, 9} are multifunctional cytokines that have well-established roles in inflammatory responses. In adipose tissue, these cytokines share a common activity in delivering potent signals for stimulation of lipolysis ^{10– 13} . Increased lipolytic activity in obese adipose tissue increases free fatty acid (FFA) flux into the circulation. From a lipocentric view, elevated plasma levels of non-esterified fatty acids (NEFA) leads to increased amounts of atherogenic lipoproteins in the circulation resulting in unmanaged hyperlipidemia ¹⁴ , often accompanied by reduced systemic insulin responsiveness ^{15, 16} .

The metabolic actions of IL-6 are diverse. Determination of which actions take precedence is largely dictated by tissue and metabolic context ¹⁷ . For example, IL-6 is an important mediator of the acute phase response which includes hepatic effects to increase glucose output and elevate CRP levels ^{17, 18} . IL-6 secretion is also increased as a result of exercise ^{19, 20} , which in turn increases glucose oxidation ²¹ and insulin sensitivity ²² in skeletal muscle. IL-6 infusion in humans increased circulating FFA levels ^{11, 23} , and when adipose tissue or isolated adipocytes were treated with IL-6, lipolytic activity was increased ¹⁷ . In adipocytes, IL-6 binds to a cell surface heterodimer composed of the IL-6 receptor and gp130 ^{24, 25} , and activates two intracellular signaling pathways; the Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway, and the p44/42 Mitogen-activated protein kinase (MAPK) pathway ^{17, 26} .

TNFα is a potent metabolic effector that, in adipocytes, signals primarily through TNFα receptor-1 ²⁷ . Intracellular signaling in adipocytes is mediated by p44/42 MAPK and Jun N-terminal kinase (JNK) ^{12, 28} . Once activated, these pathways induce phosphorylation of perilipin to recruit hormone sensitive lipase for triacylglycerol hydrolysis and the release of FFA, and to downregulate perilipin expression ^{29, 30} . Perilipin is a phosphoprotein that coats intracellular lipid droplets in adipocytes to maintain minimal lipolytic activity. Phosphorylation of perilipin serves a dual purpose: to release bound CGI-58 to activate adipose triglyceride lipase and to relocate perilipin away from the lipid droplet permitting catalytic access for activated lipases ³¹ . However, signaling the sequence of events leading to lipolytic activation through JAK/STAT, p44/42 MAPK and JNK pathways is not the normal physiologic response to increased energy demands requiring the release of fatty acid fuel stores from adipocytes. Normal physiologic activation of lipolysis by β-adrenergic and glucagon signaling during periods of increased systemic energy demands is mediated through heterotrimeric G-protein activation followed by increased intracellular cAMP and protein kinase-A (PKA) activation. Obese adipose tissue is subject to normal β-adrenergic and glucagon stimuli to regulate energy balance; however, this tissue is also subject to additional lipolytic stimuli by IL-6 and TNFα. With the presence of multiple pathways for lipolytic activation, i.e. the normal endocrine/neural pathway and cytokine-mediated pathways, it is unclear whether the effects of multiple signaling events on lipolysis are additive or coincident; that is, do IL-6 and TNFα stimulate lipolytic activities that are in excess of that provided by maximal β-adrenergic activation, or do the pathways activated by these cytokines merge into the downstream β-adrenergic pathway and are they unable to add significantly beyond maximal β-adrenergic activation. To address this question, we have measured lipolytic activity in 3T3-L1-derived adipocytes activated by a β-adrenergic agonist, IL-6 or TNFα, both individually and in combination as co- and tri-stimulation experiments.

Our primary objective for this study was to determine if cytokine stimulation (IL-6 or TNFα) heightens lipolytic activity (as measured by glycerol release) over and above what is achieved by normal β-adrenergic signaling. To obtain this quantitative evaluation, we first incubated mature 3T3-L1-derived adipocytes with varying concentrations of either isoproterenol (a β-adrenergic agonist), IL-6 or TNFα individually in order to determine the concentration of ligand that provided maximal lipolytic stimulation. This will ensure that individual ligands will be used at concentrations that maximally activate their respective signaling pathways in co- and tri-stimulation experiments. From our titration study, we have determined that maximal lipolytic stimulation for each ligand is achieved with the following concentrations: 1 µM for isoproterenol, 2 nM for IL-6 and 0.58 nM for TNFα (data not shown).

Co- and tri-stimulation experiments were performed using the ligand concentrations determined above. When adipocytes were incubated with the individual ligands, different levels of lipolytic activation were noted with IL-6 < isoproterenol < TNFα ( Figure 1. compare bar 2 < bar 1 < bar 5). These differences are likely due to activation of different signaling pathways which have varying quantitative effects on lipolytic activation. Co-stimulation of adipocytes with isoproterenol and IL-6 resulted in lipolytic activity that was greater than that achieved by stimulation with the individual ligands ( Figure 1. compare bar 3 with bars 1 and 2), suggesting parallel activation of different signaling pathways that merge into downstream lipolytic activation. The level of increased lipolytic activation from isoproterenol and IL-6 co-stimulation is approximately the sum of the individual ligands, suggesting an additive effect of contributions from two independent pathways, likely cAMP/PKA and p44/42-JAK/STAT, respectively. When isoproterenol and TNFα were combined, again an additive effect on lipolytic activation was observed ( Figure 1. compare bar 6 with bars 4 and 5), similarly suggesting a summing of the effects caused by the activation of two separate signaling pathways, cAMP/PKA and p44/42-JNK, respectively.

In vivo, obese adipose tissues express both IL-6 and TNFα when they are inflamed and susceptible to concurrent stimulation by both cytokines. To determine the effects of dual cytokine stimulation on lipolysis, adipocytes were incubated with both IL-6 and TNFα. In this case, lipolytic activation was somewhat more than an additive response ( Figure 1. compare bar 7 with bars 2 and 5), suggesting that independent, as well as overlapping, signaling pathways were activated. The independent pathways include JAK/STAT for IL-6 and JNK for TNFα, while both cytokines are capable of activating the p44/42 pathway. The greater than additive response is likely due to dual stimulation of the common p44/42 pathway. Finally, in order to simulate a physiological environment where obese, inflamed adipose tissue undergoes normal β-adrenergic stimulation during fasting and/or exercise, we treated adipocytes with a triple combination of isoproterenol, IL-6 and TNFα. This condition resulted in the highest level of lipolytic activation with a value slightly greater than adding isoproterenol stimulation to a combined treatment of IL-6 and TNFα ( Figure 1. compare bar 8 with bars 1 and 7).

The level of lipolytic activation from the triple stimulation with isoproterenol, IL-6 and TNFα far surpassed that of normal β-adrenergic stimulation alone, and provides mechanistic evidence for the cause of hyperlipidemia in obese individuals. Under normal circumstances (in lean individuals), β-adrenergic signaling is activated during fasting and exercise to mobilize fatty acids from adipose tissue and compensate for a negative systemic energy balance. Once the energy balance has returned to homeostasis, β-adrenergic stimulation is inactivated and the release of fatty acids is halted to prevent excessive plasma lipid levels. Obese individuals are also subject to normal β-adrenergic, epinephrine and norepinephrine stimulation of adipose tissue due to stress responses or negative energy balance, and this stimulation signals through the heterotrimeric G-protein, adenylyl cyclase, cAMP, PKA network ³² . In addition to this signaling, cytokines produced in inflamed obese adipose tissue also activate additional pathways that make a cumulative addition to the normal lipolytic response. Evidence provided here suggests that IL-6- and TNFα-activated pathways in adipose contribute to increased lipolysis through both independent and common signaling pathways ( Figure 2). In considering therapeutic options for obese individuals, maintaining normal β-adrenergic signaling is vital to manage routine changes in energy balance that occur due to cyclical variations in physical activity. However, the contributions of IL-6 and TNFα to increased lipolytic activity being additive to normal β-adrenergic stimulation indicates that these pathways (p44/42, JAK/STAT and JNK) represent excellent therapeutic targets that will prevent excessive lipolysis, yet minimally interfere with maintaining normal responses to varying energy demands.

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2014 Card N et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

F1000Research: Dataset 1. Glycerol release following isoproterenol, IL-6 and TNFα stimulation, 10.5256/f1000research.4151.d27711 ³³