Regulation of inducible Nitric Oxide synthase in adipocytes and Perivascular adipose tissue by the Sphingolipid system

Abstract

Obesity is a major risk factor for cardiovascular disease, often characterized by chronic inflammation that can disrupt normal metabolic processes. One key factor in this inflammatory response is the upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. The induction of iNOS in adipocytes and adipose tissue leads to increased production of NO which, in turn, contributes to the regulation of adipose tissue function and also influences lipid metabolism, insulin sensitivity, and the modulation of vascular function.

Perivascular adipose tissue (PVAT) surrounds blood vessels and has an anti- contractile effect, helping to maintain vascular homeostasis via release of a variety of bioactive molecules. In obesity-induced inflammation, some studies have demonstrated that inflammation within the PVAT can compensate or have an adaptive role to preserve endothelial dysfunction function; via upregulating iNOS protein and increasing NO production. Sphingolipid system, encompassing a complex network of bioactive lipids like ceramides, sphingosine, and sphingosine- 1-phosphate (S1P), regulates various cellular processes in adipose tissue. Although research has revealed that the sphingolipid system promotes inflammation in adipose tissue, it remains unclear how this system regulates iNOS expression and function. In response, this thesis has characterised the role of the sphingolipid system in iNOS-derived nitric oxide production in adipose tissue and addressed how this influences PVAT-mediated anticontractile activity under inflammatory conditions. The aims were achieved by using a 3T3-L1 adipocyte cell line as well as thoracic aortic PVAT from mouse and rat to study vascular function. Interleukin- 1β (IL-1β) or macrophage conditioned media were used in this study to induce iNOS expression and NO production.

In this thesis, Chapters 3 and 4 focus on the potential role of the sphingolipid system on IL-1β-induced iNOS expression and NO production in 3T3-L1 adipocytes. Initially, it was found that stimulation of 3T3-L1 adipocyte with IL-1β increases sphingosine Kinase 1 (SphK1) expression and that S1P-produced by SphKs enzymes activates various proinflammatory pathways including the phosphoinositide 3- kinase/ protein kinase B pathway (PI3K/Akt), mitogen-activated protein kinase MAPK kinase (MEK)/extracellular signal-regulated kinase pathway (MEK-ERK), Jun N-terminal kinase pathway (JNK), but apparently had no effect on the nuclear factor kappa B pathway (NF-KB). In addition, exogenous S1P augmented-IL-1β stimulated iNOS expression and NO production in 3T3-L1 adipocytes which was suggested to be through the Akt and MAPKs pathways (MEK-ERK pathway and JNK pathway). Chapter 4 investigated more fully how distinct SphKs isoforms and S1PRs modulate iNOS regulation in adipocytes stimulated with IL-1β. The iNOS expression and NO production stimulated by IL-1 β was inhibited by pre-treatment with a SphK1 inhibitor (PF543) but not a SphK2 inhibitor (ROME). Moreover, it was demonstrated that S1PR2 inhibition with JTE 013 strongly suppressed IL-1β- induced iNOS expression and NO production in 3T3-L1 adipocytes. This effect was accompanied by inhibition of MAPK and PI3K/Akt signalling, suppressing phosphorylation of ERK1/2, P38, JNK and Akt. Inhibition of ABCA1 with glybenclamide suppressed the additive effect of S1P on iNOS expression and NO production in stimulated adipocytes. Collectively, by targeting SphKs/S1P/S1PRs axis pathway, Chapter 3 and 4 provided evidence that SphK1/S1P/S1PR2/ MAPKs and PI3K/Akt axis is required for IL-1β induced iNOS expression and NO production in adipocytes.

In addition, chapter 4 has shown that conditioned medium from activated RAW 264.7 macrophages stimulated iNOS expression and NO production in 3T3-L1 adipocyte; however, prior inhibition of SphK1/S1PR2 axis attenuated this. Similarly, inhibiting S1PR2 in adipocytes prior to stimulation with conditioned medium diminishes iNOS expression and NO production.

In chapter five, it was found that S1P or S1P agonists did not modulate vascular tone in mouse thoracic aorta. Also, IL-1β did not affect the anticontractile effect of PVAT in mouse experiments and pre-treatment with S1P exerted no effect. However, in rat aorta, it was confirmed that S1P induced a vasorelaxant effect. IL-1β was found to enhance the relaxant effect of PVAT in rat aorta, which was most likely mediated by iNOS upregulation, increased NO production, and potassium channel activation in the vascular smooth muscle cells. Moreover, IL- 1β upregulated SphK1 in rat aortic PVAT; however, inhibiting SphK1/S1PR2 axis did not reverse the hyporeactivty induced by IL-1β. Collectively, IL-1β induces a hyporeactive effect in rat aortic vessels independent of the sphingolipid axis pathway.

In summary, the results of this study imply that IL-1β induces an increase in SphK1 expression in 3T3-L1 adipocytes. The increased SphK1 induced by IL-1β appears to increase S1P production from adipocytes which could contribute to IL-1β- mediated iNOS regulation through activating S1PR2. However, in the functional studies on rat aortic vessels with intact PVAT, it is likely that IL-1β-mediated hyporeactivity via iNOS upregulation was independent of the SphK1/S1P/S1PR2 axis.