Interplay between AMP-activated protein kinase (AMPK) and The Sphingolipid System in Adipose Tissue Regulation

Abstract

Introduction: Cardiovascular diseases represent a global health burden, with vascular dysfunction and chronic low-grade inflammation recognised as key pathological drivers. Published evidence highlights the crucial regulatory role of perivascular adipose tissue (PVAT) in maintaining vascular homeostasis. Dysfunctional PVAT with has been implicated in the development of vascular disease. Within this context, sphingosine kinase 1 (SphK1) and its bioactive lipid product, sphingosine-1-phosphate (S1P), is noted as a modulator of vascular tone and inflammation. Furthermore, AMP-activated protein kinase (AMPK) an enzyme which promotes cellular energy homeostasis, serves as a vascular function regulator. The interplay between AMPK signalling and the SphK1/S1P axis within PVAT represents a promising therapeutic target for ameliorating vascular dysfunction. This thesis investigates the functional interactions between AMPK, SphK1/S1P signalling within cultured adipocytes and aortic PVAT, advancing understanding of vascular biology in the hope of identifying novel therapeutic strategies for cardiovascular and metabolic diseases. Methods and Materials: This chapter outlines the experimental methodologies and analytical techniques used to investigate the interplay between AMP activated protein kinase (AMPK) and sphingosine-1-phosphate (S1P) in the regulation of adipose tissue, with a particular emphasis on the role of PVAT. Wild type (WT) and AMPKα1 knockout (KO) mice were employed as experimental models. Vascular function was assessed using wire myography to measure relaxation responses of thoracic aortic rings following exposure to S1P, AMPK activators (AICAR, A-769662, and Compound 991), and their combinations. The contribution of PVAT-derived mediators was investigated by comparing vascular responses in rings with and without intact PVAT. Adipokine secretion was analysed using a proteome-based mouse adipokine array, while nitric oxide (NO) production was quantified using a Sievers 280 NO analyser. Quantitative real-time PCR and western blotting were used to assess gene and protein expression in PVAT, mouse embryonic fibroblasts (MEFs), and 3T3-L1 adipocytes. Additionally, immunofluorescence microscopy was employed to examine the localisation of sphingosine kinase 1 (SphK1). All experiments were conducted under standardised iii laboratory conditions, and data were analysed using appropriate statistical methods to ensure accuracy, reproducibility, and scientific validity. Results: Chapter 3 explore the regulatory relationship between AMPK and SphK1 during adipocyte differentiation and in PVAT. Using 3T3-L1 adipocyte cells, mouse embryonic fibroblast (MEF) cells which lack AMPK, and mouse abdominal and thoracic PVAT, SphK1 expression was assessed in adipocytes and in cells lacking AMPK. SphK1 protein levels notably increased during adipocyte differentiation, highlighting a possible role in adipogenesis. In AMPKα1/α2 knockout MEFs, increased SphK1 protein levels were observed without corresponding changes in mRNA expression. Furthermore, tissue-specific differences were noted in PVAT, with distinct regulatory patterns observed between thoracic and abdominal PVAT. Chpter4 examine the roles of S1P and AMPK activation, using the activator AICAR, in modulating vascular function through PVAT in mouse thoracic aortic rings. S1P alone showed no vasorelaxant effects, while AICAR caused a significant vascular relaxation. S1P and AICAR combined substantially augmented this relaxation, particularly in endothelium-denuded aortic rings containing PVAT. This effect was significantly reduced in AMPKα1 knockout mice, suggesting a critical role for the AMPKα1 subunit. Further experiments suggest that increased secretion of PVAT derived adiponectin and nitric oxide could be potential mechanisms. S1PR1, a vascular-expressed receptor for S1P was identified as a key mediator of these effects. Findings highlight the complex interplay between S1P signalling, AMPK activation, and PVAT-derived factors in vascular regulation. Chapter 5 investigate the impact of S1P and different AMPK activators, namely Compound 991 (C991), and A-769662, on vascular relaxation mediated by PVAT in mouse thoracic aortic rings. As in Chapter 4, S1P alone showed no vasorelaxation effects, while each AMPK activator induced significant relaxation, particularly in PVAT-containing vessels. However, combined treatments with S1P and either C991 or A-769662 resulted in non-significant enhanced vascular relaxation, highlighting that the mechanism of AMPK activation may be crucial for this enhancement of relaxation. Discussion: Chapter 3 demonstrated that AMPKα1/α2 knockout (KO) leads to elevated SphK1 protein expression in MEFs without corresponding changes in mRNA levels, indicating post-transcriptional regulation. This deficiency also enhances iv ERK1/2 and JNK signalling, consistent with a shift toward a pro-inflammatory phenotype. Importantly, SphK1 was selectively upregulated in thoracic PVAT of AMPKα1 KO mice, but not in abdominal PVAT, underscoring a depot-specific regulatory role of AMPK. Chapter 4 demonstrated that while S1P alone exerts limited vasorelaxant effects, its combination with AICAR, an AMPK activator, significantly enhances PVAT mediated vascular relaxation through nitric oxide and adiponectin pathways. Chapter 5 explored the effects of direct AMPK activators, A-769662 and Compound 991, confirming their ability to induce vascular relaxation, although without the synergistic enhancement observed with AICAR and S1P. The pivotal role of AMPK signalling in integrating metabolic and inflammatory pathways is underscored and its potential as a therapeutic target for vascular dysfunction highlighted. Conclusion: This thesis provides novel insights into the regulatory interplay between sphingosine-1-phosphate (S1P) signalling, AMPK activation via AICAR, and the functional role of perivascular adipose tissue (PVAT) in modulating vascular relaxation in the mouse thoracic aorta. The combination of AICAR and S1P enhanced vasorelaxation, highlighting the importance of indirect AMPK activation in promoting PVAT-derived adiponectin and nitric oxide. The selective upregulation of SphK1 in AMPK-deficient tissues suggests a shift toward a pro inflammatory phenotype. These findings identify the SphK1/S1P–AMPK axis as a potential target for improving vascular health.