title: Mechanisms by which AMP-activated protein kinase in perivascular adipose tissue modulates vascular function and adipogenesis
Creators: name: Katwan, Omar Jassim
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: AMPK, adipocytes, PVAT, VSMCs, adipogenesis, VSMC contraction/relaxation signalling, adiponectin.

description: AMP-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis, which acts to redress energy imbalance via stimulation of catabolic processes and inhibition of anabolic processes. Activation of AMPK in adipocytes has multiple beneficial effects including suppression of lipogenesis, adipogenesis and pro-inflammatory signalling, modulation of thermogenesis and adiponectin secretion. Perivascular adipose tissue (PVAT) surrounds most blood vessels and also secretes factors which are reported to improve vascular reactivity under physiological conditions. In contrast, under pathophysiological conditions such as obesity, PVAT secretes factors that promote cardiovascular disease (CVD), such that dysfunctional PVAT may provide a direct mechanistic link between obesity, type 2 diabetes and increased risk of CVD. Targeting dysfunctional adipose/PVAT may therefore provide new therapeutic strategies to limit the development of obesity-related CVD. Recent work in our laboratory has demonstrated that the anti-contractile effect of PVAT on endothelium-denuded aortic rings was lost when PVAT was derived from mice lacking AMPKα1. Furthermore, this was associated with reduced adiponectin release from PVAT and phenocopied by an adiponectin receptor blocking peptide. Those previous studies also demonstrated that adiponectin phenocopied the effect of normal PVAT, yet the molecular mechanism by which PVAT and adiponectin regulate VSMC contraction/relaxation signalling and the role of AMPK in this context has not been investigated previously. The studies described in this thesis sought to determine the profile of AMPK isoform distribution in 3T3 L1 adipocytes, primary adipocytes and adipose tissue, and how this alters during adipogenesis. Additionally, the molecular mechanisms by which secreted factors from PVAT regulate contraction/relaxation signalling in human aortic VSMCs (HAoVSMCs) was investigated. AMPKα1 and β2 were the principal AMPKα and β isoforms in adipocytes in terms of the contribution to total cellular AMPK activity. Moreover, there was a shift from complexes containing β1 to those containing β2 during adipogenesis. AMPK complexes containing α1 and β2 contributed ~85% of total cellular AMPK activity in 3T3-L1 adipocytes, rodent adipocytes and human subcutaneous adipose tissue. Downregulation of AMPKβ2 but not AMPKβ1 with siRNA inhibited lipid accumulation, adiponectin production and secretion during adipogenesis in 3T3-L1 adipocytes. Furthermore a small molecule, MT47-100, selectively inhibited AMPK complexes containing β2 but not β1, inhibited lipid accumulation, adiponectin production and secretion during 3T3-L1 adipogenesis. Mechanistically, MT47-100 inhibited troglitazone-stimulated upregulation of PPARγ and C/EBPα. Conditioned medium (CM) from PVAT of wild type (WT) mice inhibited U46619- stimulated phosphorylation of myosin light chain (MLC) and cofilin, inhibited actin polymerisation in HAoVSMCs, whereas (CM) from PVAT of AMPKα1 knockout (KO) mice stimulated MLC and cofilin phosphorylation in addition to actin polymerisation, mimicking the contractile effect of U46619. CM from WT PVAT but not KO PVAT activated AMPK in HAoVSMCs. CM from KO PVAT but not WT PVAT may cause VSMC Ca2+ sensitisation but did not differentially alter HAoVSMC Ca2+ levels since CM from both genotypes inhibited U46619-stimulated intracellular Ca2+ levels. Globular adiponectin inhibited U46619-stimulated aortic ring contraction. In HAoVSMCs, adiponectin inhibited U46619-stimulated MLC phosphorylation at the sites regulated by MLC kinase (MLCK) and Rho kinase (ROCK), in addition to inhibition of myosin phosphatase target subunit 1 (MYPT1) phosphorylation, and ROCK activity, whilst tending to inhibit cofilin phosphorylation and F-actin formation. These data suggest that globular adiponectin phenocopies many of the actions of CM from PVAT of WT mice. In conclusion, these data demonstrate that AMPK has an important role in maintaining adipocyte function. In particular, the shift from AMPK complexes containing β1 to complexes containing β2 may be important for adipogenesis, and AMPKα1 plays an essential role in maintaining the anticontractile effect of PVAT likely, at least in part, through maintaining adiponectin secretion. Furthermore, adiponectin has direct actions on vascular smooth muscle to inhibit contractile signalling pathways.