AMP-activated Protein Kinase (AMPK) is a central regulator of energy homeostasis and a promising drug target for metabolic disorders. It exists as complexes of three subunits, a catalytic alpha, and two regulatory beta and gamma subunits. The regulation of AMPK involves reversible phosphorylation and allosteric regulation by adenine nucleotides. It is activated by phosphorylation of Thr172 on the catalytic alpha subunit as a consequence of various energy-depleting conditions. Once activated, AMPK regulates a plethora of metabolic processes through the phosphorylation of target proteins to maintain energy homeostasis. The beta subunit has a vital role as a structural scaffold stabilising the AMPK heterotrimeric complex. It is also known to regulate AMPK activity through different posttranslational modifications (i.e., phosphorylation and myristoylation). Although myristoylation of Glycine-2 (Gly2) of the beta subunit has been shown to be required for sensing stress signals and achieving maximum AMPK activity in vitro, its physiological relevance at the cellular and organismal levels remains unknown. Critically, the underlying molecular mechanism by which beta subunit myristoylation controls AMPK activity is elusive. The primary aim of this thesis was to investigate the molecular basis of AMPK regulation by the beta subunit myristoyl switch. I showed that mouse embryonic fibroblasts (MEFs) isolated from knock-in (KI) mice carrying Gly2 to Ala point mutation of beta1 and beta2 isoforms (beta1/2 G2A double knock-in (DKI)) displayed increased activity and phosphorylation of Thr172 in the beta subunit. Using proximity ligation assay, I found that the loss of beta1 myristoylation impedes the interaction/proximity of the phosphatases PPM1A/B with AMPK in cells. In vivo, beta1 G2A KI mice showed increased AMPK activity in the liver and were protected from high-fat diet-induced obesity, hepatic lipid accumulation, and insulin resistance. The second aim of the thesis focused on the identification of novel AMPK substrates to expand our understanding of the AMPK system/signalling in the control of metabolic and also in non-metabolic processes. We performed an unbiased phosphoproteomics analysis which revealed that AMPK phosphorylates several proteins involved in regulating Golgi structure and function. I observed that pharmacological activation of AMPK induces Golgi fragmentation in wild-type but not in AMPK-deficient human U2OS cells and MEFs. We identified AMPK-dependent phosphorylation of three Golgi-related proteins and focused on the Oxysterol-binding protein like 9 (OSBPL9), a novel AMPK substrate phosphorylated on a threonine residue (Thr335). Interestingly, knockdown of OSBPL9 in cells induced Golgi fragmentation, linking the AMPK-OSBPL9 pathway to Golgi regulation. Collectively, this study expands our understanding of the regulation and novel biological roles of AMPK. This will advance future studies to elucidate the significance of AMPK in the treatment of metabolic as well as non-metabolic disorders.