Glucagon regulates ACC activity in adipocytes through the CAMKKβ/AMPK pathway

Abstract

Glucagon is important for regulating lipid metabolism in part through its inhibition of fatty acid synthesis in adipocytes. Acetyl-CoA carboxylase 1 (ACC1) is the rate-limiting enzyme for fatty acid synthesis. Glucagon has been proposed to activate cAMP-dependent protein kinase A (PKA), which phosphorylates ACC1 to attenuate the lipogenic activity of ACC1. Because AMP-activated protein kinase (AMPK) also inhibits fatty acid synthesis by phosphorylation of ACC1, we examined the involvement of AMPK and its upstream kinase in the glucagon-elicited signaling in adipocytes in vitro and in vivo. LC-MS-MS analysis suggested that ACC1 was phosphorylated only at Ser 79 , an AMPKspecific site, in glucagon-treated adipocytes. Pharmacological inhibitors and siRNA knockdown of AMPK or PKA in adipocytes demonstrate that glucagon regulates ACC1 and ACC2 activity through AMPK but not PKA. By using Ca 2ϩ /calmodulin-dependent protein kinase kinase-␤ knockout (CaMKK␤ Ϫ/Ϫ ) mice and cultured adipocytes, we further show that glucagon activates the CaMKK␤/ AMPK/ACC cascade. Additionally, fasting increases the phosphorylation of AMPK and ACC in CaMKK␤ ϩ/ϩ but not CaMKK␤mice. These results indicate that CaMKK␤/AMPK signaling is an important molecular component in regulating lipid metabolism in adipocytes responding to glucagon and could be a therapeutic target for the dysregulation of energy storage.acetyl-coenzyme A carboxylase; Ca 2ϩ /calmodulin-dependent protein kinase kinase-␤; adenosine 5=-monophosphate-activated protein kinase; lipid metabolism; fatty acid synthesis; energy mobilization ENERGY HOMEOSTASIS AT THE WHOLE BODY LEVEL is tightly controlled by a myriad of biochemical signaling in tissues mediating metabolism, i.e., adipose tissue, muscle, liver, and hypothalamus. Regulating multiple metabolic processes, AMP-activated protein kinase (AMPK) is widely recognized as a cellular energy gauge (15). Activated AMPK upregulates catabolic pathways and downregulates anabolic pathways to promote ATP generation in peripheral tissues (27,34,35). Furthermore, orexigenic and anorexigenic signals regulate hypothalamic AMPK to modulate food intake (28). Upon energy deficiency, AMPK is phosphorylated at Thr 172 in its catalytic ␣-subunit by one of two AMPK kinases (AMPKK): Ca 2ϩ / calmodulin-dependent protein kinase kinase-␤ (CaMKK␤ or CaMKK2) or tumor suppressor LKB1. Expressed abundantly in the brain (41), CaMKK␤ controls food intake by regulating hypothalamic AMPK, leading to the production of the orexigenic hormone neuropeptide Y (4). Although many studies show that LKB1 acts as an AMPKK in the periphery (8,20,26), CaMKK␤ may still be involved in AMPK activation, which is shown by experiments using cultured adipocytes (12).Among the pantheon of hormones regulating energy mobilization, glucagon is widely thought to exert its major effects on regulating glycogenolysis and gluconeogenesis in the liver (42). However, the metabolic effects of glucagon may also involve tissues other than the liver because the glucagon receptor is expressed ...

The Ca 2+ /Calmodulin/CaMKK2 Axis: Nature's Metabolic CaMshaft

“…Although CaMKK2 is not expressed in mature adipocytes [44], studies have reported such expression in adipose exposed to exogenous hormonal stimulation [46,47]. In response to thyroid hormone, increased Ca 2+ concentration and AMPK activation via CaMKK2 was observed in adipocytes hypomorphic for Lkb1, an alternative upstream kinase for AMPK activation [47].…”

“…Meanwhile, CaMKK2 reportedly regulates AMPK action in response to glucagon, which then phosphorylates acetyl-CoA carboxylase 1 (ACC1) in adipocytes. Glucagon-induced phosphorylation of ACC1 attenuates its activity during FA synthesis [46]. Since increased intracellular Ca 2+ within adipocytes was not induced by glucagon, the mechanism for AMPK activation remains to be clarified and reconciled with previous findings.…”

Physiological and Pathophysiological Control of Glucagon Secretion by Pancreatic α-Cells

“…Experiments on cell lines expressing the glucagon receptor have shown that stimulation of IP 3 production requires high glucagon concentrations (Xu and Xie 2009). Through mechanisms that remain incompletely understood, glucagon also activates AMP-activated protein kinase (AMPK) via Ca 2+ /calmodulin-dependent protein kinase kinase-β (CaMKKβ or CaMKK2) (Kimball et al 2004;Peng et al 2012) and p38 MAPK (Cao et al 2005), and it induces acetylation of Foxa2, an activator of lipid metabolism and ketogenesis (von Meyenn et al 2013).…”

“…It is therefore more easily observed in conditions of insulinopenia, and it is not clear whether or not it stimulates lipolysis under normal physiological conditions in human (Ranganath et al 2001;Arafat et al 2013;Gravholt et al 2001). Glucagon also inhibits fatty acid synthesis via an AMPK-dependent inactivating phosphosphorylation of ACC in adipocytes (Peng et al 2012).…”

Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages

“…hmCD40-RAW 264.7 cells were transfected with JNK1/2 small interfering RNA (siRNA) (Dharmacon), ULK1 siRNA (Life Technologies), CaMKK␤ siRNA (27), AMPK␣1 siRNA (27), AMPK␣2 siRNA (27), or control siRNA by using an Amaxa Nucleofector kit. Cells were subsequently transfected with a plasmid encoding tandem monomeric red fluorescent protein (RFP)-green fluorescent protein (GFP)-tagged LC3 (tfLC3) (28) (gift from T. Yoshimori, National Institute for Basic Biology, Okazaki, Japan).…”