Adiponectin Signaling: The Calcium Connection: R&D Systems

Adiponectin, also known as Acrp30, is an adipocytokine that positively regulates glucose and lipid metabolism by increasing insulin sensitivity, stimulating fatty acid oxidation and glucose uptake, and suppressing hepatic glucose production. Adiponectin deficiency is often associated with obesity and is highly correlated with insulin resistance, mitochondrial dysfunction, and dyslipidemia.^1,² Over the past 15 years, a significant amount of research has been dedicated to determining the mechanisms by which Adiponectin functions. In 2003, Yamauchi et al. identified two Adiponectin receptors, Adipo R1 and Adipo R2.³ While subsequent data from this group and others have clearly shown that these receptors bind Adiponectin and mediate its metabolic effects, details of how they do so have remained unclear.^2,^3,^4,⁵

A recently published paper from the group that originally cloned Adipo R1 and R2 now takes a significant step toward elucidating the Adipo R1 signaling pathway.⁶ This was accomplished using mice in which Adipo R1 was specifically deleted in skeletal muscle (muscle-Adipo R1KO). Adipo R1 is the primary Adiponectin receptor expressed in skeletal muscle, the major glucose-utilizing tissue in the body.^2,^3,⁴

In vivo data from muscle-Adipo R1KO mice confirm the involvement of Adipo R1 in glucose tolerance and insulin sensitivity. This was demonstrated both by the presence of notably higher plasma glucose and insulin levels in fed muscle-Adipo R1KO mice compared to wild-type mice, and by significant changes in insulin-stimulated phosphorylation of signaling molecules such as IRS-1, Akt, p70 S6 kinase, and JNK.⁶ Muscle-Adipo R1KO mice also displayed diminished mitochondrial biogenesis, as shown by decreased activity of the transcription factor PGC1 alpha and reduced quantities of mitochondria-specific proteins and DNA.^2,⁶ As often accompanies insulin resistance, fatty acid oxidation was impaired, and oxidative stress was increased in the absence of skeletal muscle Adipo R1. Interestingly, exercise was able to improve these metabolic pathways, even in the absence of muscle Adipo R1, indicating that exercise can partially compensate for inadequate Adiponectin signaling.⁶

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Adiponectin Signaling Promotes the Activation of PGC1 alpha and Mitochondrial Biogenesis. Adiponectin binding to Adipo R1 causes a calcium influx in skeletal muscle that activates CaMKK beta. Activated CaMKK beta induces the expression and activation of PGC1 alpha via a cascade involving the activation of AMPK and increased production of the SIRT1 deacetylase. AMPK phosphorylation and SIRT1 deacetylation of PGC1 alpha increase its transcriptional activity, leading to increased mitochondrial biogenesis.

To determine the signaling defects responsible for the phenotypic changes in muscle-Adipo R1KO mice, thorough in vitro studies were performed using small interfering RNAs (siRNAs), and specific inhibitors to knock down the expression, or function, of metabolic regulatory molecules, such as Adipo R1 and R2, AMPK alpha 1 and AMPK alpha 2, CAMKK beta, PGC1 alpha, SIRT1, and LKB1 in normal mouse myocytes.⁶ Inhibition of Adipo R1, CAMKK beta, PGC1 alpha, or both AMPK alpha 1 and AMPK alpha 2 reduced Adiponectin-induced mitochondrial biogenesis. In addition, Adipo R1 and CAMKK beta siRNA suppressed the increase in PGC1 alpha expression that was induced by Adiponectin in normal mouse myocytes. Notably, both the suppression of Adipo R1 expression by siRNA, and the deletion of Adipo R1 in muscle-Adipo R1KO mice led to a defect in the influx of extracellular calcium that was observed in normal myocytes following Adiponectin treatment. This Adiponectin-mediated calcium influx is required for CaMKK beta activation, which in turn affects PGC1 alpha through multiple pathways. First, activated CaMKK beta increases the expression of PGC1 alpha, and second, it phosphorylates and activates AMPK. Activated AMPK increases the cellular NAD⁺/NADH ratio, leading to activation of the SIRT1 deacetylase.^6,⁷ Both phosphorylation of PGC1 alpha by AMPK and its deacetylation by SIRT1 enhance the transcriptional activity of PGC1 alpha, which is essential for the expression of proteins involved in mitochondrial biogenesis.^6,⁸

While these results shed new light on the cellular events associated with Adiponectin/Adipo R1 signaling, how Adipo R1 induces an influx of extracellular calcium still needs to be resolved. Four molecules are known to bind the Adipo R1 intracellular domain, including the adaptor proteins RACK1 and APPL1, the kinase regulatory subunit CK2 beta, and the endoplasmic reticulum protein ERp46. Although RACK1, CK2 beta, and ERp46 are all proposed to modulate Adiponectin signaling, their influence on calcium influx is unknown.^9,^10,¹¹ APPL1 is known to enhance signaling by Akt, a kinase required for the insulin signaling pathway, but this effect is thought to be independent of CaMKK.⁵ TRPC3, the skeletal muscle T-tubule cation channel has been shown to modulate insulin-mediated glucose uptake, but connections with Adipo R1 signals are unknown.¹² Identifying the critical intracellular Adiponectin binding proteins that connect Adipo R1 to the calcium influx is one of the final steps required for a clear understanding of the Adiponectin signaling pathway in skeletal muscle.