Adrenergic Regulation of AMP-activated Protein Kinase in Brown Adipose Tissue in Vivo

Abstract

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK ␣2 protein and activity were reduced by 45%. In ␤ 1,2,3 -adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr 172 phosphorylation. In contrast, acute ␣-or ␤-adrenergic blockade in WT mice resulted in increased AMPK ␣ Thr 172 phosphorylation and AMPK ␣1 and ␣2 activity in BAT. But short term blockade of ␣-adrenergic signaling in ␤ 1,2,3 -adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser 485/491 , a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser 485/491 phosphorylation resulting from ␣-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of ␣-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr 172 phosphorylation and chronically through changes in the ␣ catalytic subunit protein levels. Furthermore, AMPK ␣ Ser 485/491 phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects. AMP-activated protein kinase (AMPK),4 an evolutionarily conserved serine threonine kinase, is a fuel-sensing enzyme complex activated by cellular stresses that increase AMP or deplete ATP including hypoxia, ischemia, glucose deprivation, uncouplers of oxidative phosphorylation, exercise, and muscle contraction (1, 2). Once activated, AMPK phosphorylates multiple downstream substrates that function to preserve the AMP: ATP ratio through inhibition of ATP-catabolizing pathways and promotion of ATP-generating pathways. Mechanisms involved in AMPK activation include 1) binding of AMP to an allosteric site on the ␥ subunit, which renders the holoenzyme resistant to inactivating serine phosphatases and may also have direct allosteric effects on kinase activity (1, 2) and 2) phosphorylation by upstream AMPK kinases of the ␣ (catalytic) subunits on Thr 172 , which is essential for kinase activity. Recent studies employing INS-1 cells (3), hepatitis C virus harboring replicon cells (4), and isolated heart preparations (5) have demonstrated another potential regulatory mechanism; that is, AMPK may also undergo inhibitory phosphorylation on serine 485 and 491 residues of the ␣1 and ␣2 catalytic subunits, respectively. Some data suggest that upstr...

Low Concentrations of Metformin Suppress Glucose Production in Hepatocytes through AMP-activated Protein Kinase (AMPK)*

“…␤-adrenergic receptor decreased AMPK␣ phosphorylation at Thr-172 and AMPK activity in adipose tissue and cardiomyocytes (31)(32)(33). In light of these data, we reasoned that cAMP might antagonize AMPK activation by metformin, and this might be important for clinical metformin usage because diabetic patients often have hyperglucagonemia.…”

Augmenting energy expenditure by mitochondrial uncoupling: a role of AMP-activated protein kinase

“…AMPK protein amount and activity was found to be two to threefold higher in BAT than in liver (Mulligan et al 2007). Chronic cold exposure of mice led to a progressive increase in AMPK activity in BAT (Mulligan et al 2007) and prolonged lack of adrenergic stimulation led to decreased AMPK activity, which was secondary to reduction in both a1-and a2-AMPK subunits levels (Pulinilkunnil et al 2011). On the other hand, acute blockage of a-adrenergic signaling resulted in stimulation of AMPK activity, mediated by alteration in AMPK a Thr 172 phosphorylation, which could be secondary to diminished oxygen consumption in BAT (Pulinilkunnil et al 2011).…”

“…Chronic cold exposure of mice led to a progressive increase in AMPK activity in BAT (Mulligan et al 2007) and prolonged lack of adrenergic stimulation led to decreased AMPK activity, which was secondary to reduction in both a1-and a2-AMPK subunits levels (Pulinilkunnil et al 2011). On the other hand, acute blockage of a-adrenergic signaling resulted in stimulation of AMPK activity, mediated by alteration in AMPK a Thr 172 phosphorylation, which could be secondary to diminished oxygen consumption in BAT (Pulinilkunnil et al 2011). Acute stimulation of b-adrenergic signaling in mice also resulted in increased AMPK activity in BAT, leading to increased glucose uptake with both effects abolished in UCP1 knock-out mice (Inokuma et al 2005).…”

“…Thus, the mechanistic link between UCP1 and AMPK in BAT, as well as the role of AMPK in the BAT thermogenic function, remains to be clarified. Emerging evidence suggests that a2-but not a1-AMPK subunit might be required for thermogenesis in BAT (Pulinilkunnil et al 2011;Bauwens et al 2011). In addition to its potential role to regulate BAT metabolism, AMPK-mTOR signaling may promote differentiation of BAT cells (Vila-Bedmar et al 2010).…”

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“…Although AMPK is present in different tissues and organs, the subunit composition varies, and changes in cell physiology can also alter the profile of expressed subunits (Mahlapuu et al, 2004;Pulinilkunnil et al, 2011;Putman et al, 2007;Quentin et al, 2011;Stapleton et al, 1996;Turnley et al, 1999). Of particular importance at the cellular, organ and organismal level is the ability of AMPK to switch from anabolic to catabolic processes when energy supplies are low.…”