Systemic insulin sensitivity is regulated by GPS2 inhibition of AKT ubiquitination and activation in adipose tissue

Abstract

ObjectiveInsulin signaling plays a unique role in the regulation of energy homeostasis and the impairment of insulin action is associated with altered lipid metabolism, obesity, and Type 2 Diabetes. The main aim of this study was to provide further insight into the regulatory mechanisms governing the insulin signaling pathway by investigating the role of non-proteolytic ubiquitination in insulin-mediated activation of AKT.MethodsThe molecular mechanism of AKT regulation through ubiquitination is first dissected in vitro in 3T3-L1 preadipocytes and then validated in vivo using mice with adipo-specific deletion of GPS2, an endogenous inhibitor of Ubc13 activity (GPS2-AKO mice).ResultsOur results indicate that K63 ubiquitination is a critical component of AKT activation in the insulin signaling pathway and that counter-regulation of this step is provided by GPS2 preventing AKT ubiquitination through inhibition of Ubc13 enzymatic activity. Removal of this negative checkpoint, through GPS2 downregulation or genetic deletion, results in sustained activation of insulin signaling both in vitro and in vivo. As a result, the balance between lipid accumulation and utilization is shifted toward storage in the adipose tissue and GPS2-AKO mice become obese under normal laboratory chow diet. However, the adipose tissue of GPS2-AKO mice is not inflamed, the levels of circulating adiponectin are elevated, and systemic insulin sensitivity is overall improved.ConclusionsOur findings characterize a novel layer of regulation of the insulin signaling pathway based on non-proteolytic ubiquitination of AKT and define GPS2 as a previously unrecognized component of the insulin signaling cascade. In accordance with this role, we have shown that GPS2 presence in adipocytes modulates systemic metabolism by restricting the activation of insulin signaling during the fasted state, whereas in absence of GPS2, the adipose tissue is more efficient at lipid storage, and obesity becomes uncoupled from inflammation and insulin resistance.

G protein pathway suppressor 2 (GPS2) links inflammation and cholesterol efflux by controlling lipopolysaccharide‐induced ATP‐binding cassette transporter A1 expression in macrophages

“…In addition to possible chromatin differences, the GPS2-interacting set of TFs that define gene-specific effects remain yet to be clarified. Notably, GPS2 was first identified as an inhibitor of TNF-a activation (27), consistent with subsequently demonstrated nongenomic GPS2 actions in the cytoplasm (45,46). As we show here ChIPseq data clearly demonstrate nuclear chromatin-binding of GPS2 and p65 (e.g., at the Abca1 locus).…”

GPS2 regulates mitochondria biogenesis via mitochondrial retrograde signaling and chromatin remodeling of nuclear-encoded mitochondrial genes

“…Intriguingly, it does so by preventing the accumulation of H3K9me3, which could be inhibitory against H3K9ac, a mark of active transcription deposited by Gcn5/PCAF, the catalytic subunit of the SAGA complex. In addition, Rtg2-mediated regulation of Rtg1-3 nuclear import via modulation of an inhibitory phosphorylation cascade closely resembles GPS2-mediated regulation of NFkB activity, as well as our recent finding of GPS2 promoting FOXO1 stabilization and its nuclear functions via inhibition of AKT ubiquitination and activation Cederquist et al, 2017;Lentucci et al, 2016). Lastly, our in vivo data indicate that GPS2 plays a critical role in the regulation of lipid metabolism and energy expenditure in the adipose tissue, just as RTG2 has proven to be an essential factor for the regulation of nutrient metabolism in yeast cells.…”

“…To elucidate the specificity of this regulatory strategy, we next asked what constituted the molecular mechanism of GPS2-mediated regulation of transcription in the context of neMITO genes. As GPS2 is a potent inhibitor of the ubiquitin conjugating enzyme Ubc13 and regulates the expression of selected PPARγ target promoters through stabilization of an H3K9 demethylase Cardamone et al, 2014;Cederquist et al, 2017;Lentucci et al, 2017), we first investigated whether H3K9 demethylation and local ubiquitination are involved in the regulation of mitochondrial genes. This proved correct as in 3T3-L1 GPS2-KD cells we detected: i) a significant increase in the level of nonproteolytic ubiquitin chains on the promoters of mitochondrial genes Ndufv1 and Tomm20 by ChIP using an antibody specific for K63 ubiquitin chains (Fig.…”

“…Furthermore, in addition to the nuclear functions, we recently reported a number of distinct, but possibly coordinated, functions for GPS2 in the cytosol as regulator of insulin signaling and pro-inflammatory response pathways Cederquist et al, 2017;Lentucci et al, 2016). While the molecular mechanisms underlying GPS2 genomic and non-genomic functions have not been fully elucidated, our work suggests that both nuclear and cytosolic functions are linked to GPS2-mediated inhibition of lysine 63 (K63) ubiquitination events Cardamone et al, 2014;Cederquist et al, 2017;Lentucci et al, 2016). This raises a number of important questions regarding GPS2 function and regulation in the cell.…”

Subcellular origin of mitochondrial DNA deletions in human skeletal muscle

“…Antibodies against PGC1α (Millipore Ab3243), TFAM (Abcam Ab119684), NEF2L2 (Abcam ab31163), ClpP (Sigma HPA010649), Htra2 (R&D Systems AF1458), mtHsp70 (Thermofisher), Hsp60 (BD Transduction Systems 611562), GPS2 21, p62 (Progen GP62-C), Beclin1 (Millipore Ab15417), Pink1 (Abcam Ab23707), LC3-II (cell signaling 4108) and Parkin (Santa Cruz biotechnologies sc-32282) to assess signaling, were used in combination with MTCOI, SDHA and DAPI to assess changes with COX-deficiency in a single section of muscle. Antibodies that did not produce a sufficient signal-to-noise ratio, or were highly correlated with mitochondrial mass, were removed from further analysis.…”