title: The role of AMPK in the regulation of nitric oxide synthesis by perivascular adipose tissue
Creators: name: Hwej, Abdmajid Saad
Publisher: name: University of Glasgow
Year: 2023

description: Nitric oxide (NO) is a key signalling molecule in the cardiovascular, genitourinary, respiratory, nervous, and gastrointestinal systems. NO is released by the endothelium of blood vessels and inhibits vascular smooth muscle cell (VSMC) migration and proliferation, modulates vascular tone and maintains cardiovascular homeostasis. Several clinical studies have shown that a reduction of NO bioavailability has a significant role in the development of endothelial dysfunction and other cardiovascular diseases. Perivascular adipose tissue (PVAT) is the outer layer of connective tissue that surrounds most systemic blood vessels, adjacent to the adventitia. Adipose tissue provides a protective layer of tissue surrounding most of the body organs and, in the case of PVAT, regulates vascular function by production of many autocrine and paracrine molecules including adiponectin, prostacyclin and angiotensin 1–7. AMP-activated protein kinase (AMPK) plays a major role in sensing cellular energy status and signalling this information back to the mitochondria to modulate their function according to the energy demands of the cell. Previous work in our laboratory has demonstrated that the presence of PVAT enhances vascular relaxation to cromakalim in endothelium-intact thoracic aortic rings from wild type (WT), but not in mice lacking AMPKα1. Compared to previous studies which examined the role of AMPK in regulation of NO release from the endothelium, the current research has focused on the mechanisms by which AMPK regulates NO production by PVAT and what effect this has on vascular tone. In this project experiments were conducted using wild type (WT) and global AMPKα1 knockout (KO) mice. Thoracic and abdominal aortic PVAT was utilised to measure the difference in NO production between fat depots. The anti-contractile effect of PVAT from WT and KO aortic rings was studied using wire myography. Furthermore, immunoprecipitation was used to measure the Cav-1/eNOS coupling and immunofluorescence was used to study Cav-1/eNOS colocalization in 3T3-L1 adipocytes. The results showed that there was a significant decrease in NO production in conditioned media derived from both the thoracic and abdominal PVAT from KO mice compared to WT mice, although the quantities of NO generated by the abdominal aortic PVAT was much lower. Overall, eNOS activity in WT thoracic PVAT was significantly increased compared to WT abdominal PVAT suggesting that changes in eNOS activity could account for the difference in NO production. Furthermore, in abdominal PVAT, considerably more caveolin-1 (Cav-1), a negative regulator of eNOS, was detected in both WT and KO mice compared with thoracic PVAT, which may also limit NO production by abdominal PVAT. Cav-1 was detected in eNOS immunoprecipitates and levels of Cav-1/eNOS association were increased in abdominal PVAT relative to thoracic PVAT of WT mice and the total Cav-1 levels in abdominal PVAT lysates was increased. Intriguingly, Cav-1/eNOS association was significantly increased in thoracic PVAT from KO mice compared with WT mice, which might account for the reduction in NO production in KO thoracic PVAT compared with WT. In 3T3-L1 adipocyte adipogenesis, eNOS levels and NO production were significantly reduced. Incubation of 3T3-L1 preadipocytes or adipocytes with methyl-β-cyclodextrin (MβCD), which disrupts Cav-1 and therefore leading to a noticeable reduction in Cav-1/eNOS colocalization and a significant increase in NO production both in preadipocytes and adipocytes compared with non-treated cells. Incubation of 3T3-L1 adipocytes with a mutant cell–permeable scaffolding domain peptide of Cav-1 (CAV-AP) reduced Cav-1/eNOS colocalization compared with control. In functional experiments, CAV-AP reduced PE-induced contraction in both WT and KO thoracic aortic rings lacking endothelium. Overall, the results presented in this thesis demonstrate that AMPKα1 has an important role in regulating NO production by PVAT, likely through regulating Cav-1/eNOS binding. Manipulating Cav-1/eNOS binding in adipocytes using scaffolding mutant peptides without interfering with the other biological effects of endogenous Cav-1 may have beneficial effects in restoration of NO production under conditions or diseases associated with impaired NO bioavailability.