Adiponectin Activates AMP-activated Protein Kinase in Muscle Cells via APPL1/LKB1-dependent and Phospholipase C/Ca2+/Ca2+/Calmodulin-dependent Protein Kinase Kinase-dependent Pathways

The binding of the adaptor protein APPL1 to adiponectin receptors is necessary for adiponectin-induced AMP-activated protein kinase (AMPK) activation in muscle, yet the underlying molecular mechanism remains unknown. Here we show that in muscle cells adiponectin and metformin induce AMPK activation by promoting APPL1-dependent LKB1 cytosolic translocation. APPL1 mediates adiponectin signaling by directly interacting with adiponectin receptors and enhances LKB1 cytosolic localization by anchoring this kinase in the cytosol. Adiponectin also activates another AMPK upstream kinase Ca²⁺/calmodulin-dependent protein kinase kinase by activating phospholipase C and subsequently inducing Ca²⁺ release from the endoplasmic reticulum, which plays a minor role in AMPK activation. Our results show that in muscle cells adiponectin is able to activate AMPK via two distinct mechanisms as follows: a major pathway (the APPL1/LKB1-dependent pathway) that promotes the cytosolic localization of LKB1 and a minor pathway (the phospholipase C/Ca²⁺/Ca²⁺/calmodulin-dependent protein kinase kinase-dependent pathway) that stimulates Ca²⁺ release from intracellular stores.Adiponectin, an adipokine abundantly expressed in adipose tissue, exhibits anti-diabetic, anti-inflammatory, and anti-atherogenic properties and hence is a potential therapeutic target for various metabolic diseases (1–3). The beneficial effects of adiponectin are mediated through the direct interaction of adiponectin with its cell surface receptors, AdipoR1 and AdipoR2 (4, 5). Adiponectin increases fatty acid oxidation and glucose uptake in muscle cells by activating AMP-activated protein kinase (AMPK)³ (4, 6), which depends on the interaction of AdipoR1 with the adaptor protein APPL1 (Adaptor protein containing Pleckstrin homology domain, Phosphotyrosine binding domain, and Leucine zipper motif) (5). However, the underlying mechanisms by which APPL1 mediates adiponectin signaling to AMPK activation and other downstream targets remain unclear.AMPK is a serine/threonine protein kinase that acts as a master sensor of cellular energy balance in mammalian cells by regulating glucose and lipid metabolism (7, 8). AMPK is composed of a catalytic α subunit and two noncatalytic regulatory subunits, β and γ. The NH₂-terminal catalytic domain of the AMPKα subunit is highly conserved and contains the activating phosphorylation site (Thr¹⁷²) (9). Two AMPK variants, α1 and α2, exist in mammalian cells that show different localization patterns. AMPKα1 subunit is localized in non-nuclear fractions, whereas the AMPKα2 subunit is found in both nucleus and non-nuclear fractions (10). Biochemical regulation of AMPK activation occurs through various mechanisms. An increase in AMP level stimulates the binding of AMP to the γ subunit, which induces a conformational change in the AMPK heterotrimer and results in AMPK activation (11). Studies have shown that the increase in AMPK activity is not solely via AMP-dependent conformational change, rather via phosphorylation by upstream kinases, LKB1 and CaMKK. Dephosphorylation by protein phosphatases is also important in regulating the activity of AMPK (12).LKB1 has been considered as a constitutively active serine/threonine protein kinase that is ubiquitously expressed in all tissues (13, 14). Under conditions of high cellular energy stress, LKB1 acts as the primary AMPK kinase through an AMP-dependent mechanism (15–17). Under normal physiological conditions, LKB1 is predominantly localized in the nucleus. LKB1 is translocated to the cytosol, either by forming a heterotrimeric complex with Ste20-related adaptor protein (STRADα/β) and mouse protein 25 (MO25α/β) or by associating with an LKB1-interacting protein (LIP1), to exert its biological function (18–22). Although LKB1 has been shown to mediate contraction- and adiponectin-induced activation of AMPK in muscle cells, the underlying molecular mechanisms remain elusive (15, 23).CaMKK is another upstream kinase of AMPK, which shows considerable sequence and structural homology with LKB1 (24–26). The two isoforms of CaMKK, CaMKKα and CaMKKβ, encoded by two distinct genes, share ∼70% homology at the amino acid sequence level and exhibit a wide expression in rodent tissues, including skeletal muscle (27–34). Unlike LKB1, AMPK phosphorylation mediated by CaMKKs is independent of AMP and is dependent only on Ca²⁺/calmodulin (35). Hence, it is possible that an LKB1-independent activation of AMPK by CaMKK exists in muscle cells. However, whether and how adiponectin stimulates this pathway in muscle cells are not known.In this study, we demonstrate that in muscle cells adiponectin induces an APPL1-dependent LKB1 translocation from the nucleus to the cytosol, leading to increased AMPK activation. Adiponectin also activates CaMKK by stimulating intracellular Ca²⁺ release via the PLC-dependent mechanism, which plays a minor role in activation of AMPK. Taken together, our results demonstrate that enhanced cytosolic localization of LKB1 and Ca²⁺-induced activation of CaMKK are the mechanisms underlying adiponectin-stimulated AMPK activation in muscle cells.