Researchers at Yale have identified a molecule that plays a key role in the body’s inflammatory response to overeating, which can lead to obesity, diabetes, and other metabolic diseases.
The finding suggests that the molecule could be a promising therapeutic target to control this inflammation and keep metabolic diseases in check.
The study appears on June 29 in the Proceedings of the National Academy of Sciences.
When a person overeats, the body stores excess calories in the form of fat in the adipose tissue, or body fat, said lead author Xiaoyong Yang of Yale School of Medicine. As the amount of calories consumed continues to increase, this leads to inflammation in adipose tissue and the release of fatty acids into other tissues, including the liver and muscles.
“This is dangerous,” Yang said, “and leads to metabolic disorders like diabetes.”
Researchers were aware that overeating led to inflammation and metabolic diseases, but until now, they did not know the precise way that the body’s immune cells, such as macrophages – which react to excess calorie consumption – contributed to this process.
The new research by Yang and team zeroed in on a pathway called O-GIcNAc signaling, which activates when a person overeats, instructing the cells to limit inflammation.
Inflammation happens when the body’s immune system reacts to injury or threat, and involves increased blood flow, capillary dilation, and an influx of white blood cells.
“The body is smart,” said Yang, associate professor of comparative medicine and of cellular & molecular physiology. “It tries to protect against inflammation when fat builds up in the body. We discovered a key pathway that quenches inflammation caused by overnutrition.”
In particular, the researchers found that OGT (O-GIcNAc transferase), an enzyme that activates GIcNAc signaling, was responsible for activating the body’s pro-inflammatory response by turning on or off a specific signaling pathway in macrophages.
“The macrophage can be a good guy or a bad guy,” Yang said. “It becomes a bad guy in overnutrition, secreting a lot of inflammatory factors. In other contexts, it’s a good guy and has an anti-inflammatory effect. We found out that OGT tries to stop the macrophage from becoming a bad guy—to stop the pro-inflammatory response.”
Their finding suggests that OGT could be a target for new therapies to suppress inflammation and improve health.
The study also sheds light on the workings of glutamine and glucosamine, nutritional supplements recommended by doctors for arthritis and inflammation of the joints, Yang said. While researchers have known that these supplements promote O-GlcNAc signaling and reduce inflammation, they did not know how this process worked.
“Our finding further implicates how glutamine and glucosamine suppress inflammation,” Yang said.
Reprogramming of cellular metabolic activities has recently been demonstrated to play a critical role in the activation of the immune system and hyperinflammation (Buck et al., 2017, O’Neill et al., 2016). Increased glucose uptake and glycolysis occur in classically activated innate immune cells in vitro and in vivo (Everts et al., 2014).
A widely accepted concept in the immunometabolism research field is that elevated catabolic activity in activated immune cells is necessary for meeting the increased demand of biomolecules and energy for effective immune functions.
Those functions (including cell migration, phagocytosis, and cytokine production) are necessary for host response against invading pathogens or tissue injury during inflammation. Recent progress has broadened our understanding of how metabolic reprogramming modulates immune functions in multiple aspects.
For example, a variety of metabolic enzymes involved in the glycolysis and mitochondrial metabolic pathways have been identified as playing essential roles in affecting innate immune cell function (O’Neill et al., 2016).
Moreover, many intermediate metabolites, such as succinate (Tannahill et al., 2013), fumarate (Arts et al., 2016), itaconate (Bambouskova et al., 2018, Mills et al., 2018), and α-ketoglutarate (Liu et al., 2017), have recently been reported to participate in immune activation or modulation.
Therefore, the metabolic system regulates immune cell function and inflammation through combined strategies.
Glucose serves as a major nutrient to fuel cellular metabolic activities.
Three major glucose metabolic pathways, namely glycolysis, the pentose phosphate pathway (PPP), and the hexosamine biosynthesis pathway (HBP), collaboratively determine how glucose is processed.
HBP is a unique glucose metabolism pathway leading to the generation of its end product uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which is further utilized by the O-GlcNAc transferase (OGT) for protein modification, namely O-GlcNAcylation (Levine and Walker, 2016).
Many proteins involved in various fundamental biological processes, including transcription factors, kinases, and enzymes, have been identified as O-GlcNAcylation targets (Yang and Qian, 2017).
Recent studies have identified several molecules involved in the innate immune signaling as O-GlcNAcylation targets. For example, O-GlcNAcylation of IKKβ, NF-κB p65, c-Rel, and TAB1 enhances their activities and promotes the transcription of NF-κB target genes. (Ozcan et al., 2010, Pathak et al., 2012, Yang et al., 2015c). We recently identified O-GlcNAcylation of the transcription factor STAT3 as an important mechanism antagonizing its activation (Li et al., 2017).
However, the specific role of OGT in innate immune function and inflammation remains poorly defined.
RIPK3 is a member of the receptor-interacting protein (RIP) family of serine/threonine kinases and contains an N-terminal kinase domain and a C-terminal RIP homotypic interaction motif (RHIM) (Silke et al., 2015).
Through RHIM-mediated protein interaction, RIPK3 forms a necrosome complex with RIPK1, and this complex is required for the induction of necroptosis, an inflammatory form of cell death (Galluzzi et al., 2017, Weinlich et al., 2017). Both RHIM and kinase activity of RIPK3 are essential for activation of downstream effector protein MLKL and execution of necroptosis (Wallach et al., 2016).
In addition to having a central role in necroptosis, elevated RIPK3 activation has been shown to promote inflammatory responses in both cell-death-dependent and -independent manners (Alvarez-Diaz et al., 2016, Moriwaki et al., 2017, Najjar et al., 2016).
Despite being a well-studied signaling pathway leading to necrosome formation, the intrinsic mechanism modulating RIPK3 activation is not well understood. In this study, we identified OGT-mediated RIPK3 O-GlcNAcylation at T467 as a key mechanism to block RHIM-mediated RIPK3-RIPK1 and RIPK3-RIPK3 interaction. Removal of OGT or RIPK3 O-GlcNAcylation promoted macrophage inflammatory response and necroptosis, both of which are dependent on RIPK3 RHIM domain and kinase activity.
As a result, genetic deletion of Ogt in myeloid cells markedly exacerbated cytokine storm and host mortality in experimental sepsis. Therefore, our findings demonstrate a check mechanism against overzealous innate immune activation through OGT-mediated RIPK3 O-GlcNAcylation.
Discussion
Previous studies have provided biochemical evidence to support the notion that O-GlcNAc signaling promotes inflammation. Several key molecules in the TLR-NF-κB signaling pathway have been identified to be O-GlcNAcylated. O-GlcNAcylation of these proteins has been shown to enhance their functional activities and promote the transcription of NF-κB target genes (Ozcan et al., 2010).
Our recent study also observed an enhanced inflammatory response and disease severity in chemically induced colitis as a result of attenuated STAT3-IL-10 signaling when OGT expression and enzymatic activity were upregulated in myeloid cells (Li et al., 2017).
In this study, we demonstrated an unexpected inhibitory effect of OGT on innate immune activation and necroptosis signaling through RIPK3 O-GlcNAcylation. Therefore, the net effect of OGT-mediated O-GlcNAc signaling in the immune system and inflammation seems to be multifaceted because of the involvement of a variety of target proteins in different immune signaling pathways.
Both the increase and decrease in OGT-mediated protein O-GlcNAcylation resulted in a similar hyperinflammatory response through distinct mechanisms. On the one hand, elevated O-GlcNAc signaling promoted activation of the innate immune cells by increasing NF-κB signaling, as well as counteracting the anti-inflammatory STAT3-IL-10 signaling.
On the other hand, loss of O-GlcNAc signaling removed an inhibitory mechanism of RIPK3 activation and consequently led to enhanced inflammatory responses and inflammation-associated necroptosis, despite increased IL-10 production.
We therefore propose that loss of homeostasis in O-GlcNAc signaling, instead of a simple one-way increase or decrease, is an important metabolic mechanism underlying the pathogenesis of inflammatory diseases.
The aforementioned studies raise an important question on how to regulate OGT-mediated O-GlcNAc signaling under pathophysiological conditions. Previous studies have shed light on two important mechanisms: controlling UDP-GlcNAc availability through regulation of HBP activity and targeting OGT gene transcription and protein degradation.
First, the availability of UDP-GlcNAc produced through HBP metabolic processes is an important determinant of OGT enzymatic activity (Hart et al., 2011). The first and rate-limiting enzyme in HBP, glutamine fructose-6-phosphate transaminase (GFPT), represents a key mediator in affecting downstream OGT signaling by determining UDP-GlcNAc concentration.
A recent study reported that GFPT is transcriptionally upregulated under endoplasmic reticulum (ER) stress, and this upregulation leads to increased protein O-GlcNAcylation and provides a protective effect against ischemia-reperfusion injury in the heart (Wang et al., 2014).
Our study observed decreased HBP activity and protein O-GlcNAcylation by TLR4 activation, despite increased glycolysis and PPP metabolic activities. In contrast, in a parallel study, we observed increased HBP flux activity and protein O-GlcNAcylation when retinoic-acid-inducible gene I (RIG-I) was activated by intracellular RNA ligands (Li et al., 2018).
The opposite O-GlcNAc signaling changes due to activation of distinct innate immune sensors highlight that GFPT-mediated HBP activity is an important mechanism regulating OGT function. How GFPT-controlled HBP activity is differentially regulated during activation of distinct pathogen-recognition-receptor signaling requires further investigation.
Second, the abundance of OGT protein is negatively regulated by a ubiquitination-mediated protein-degradation process (Yang et al., 2015b), whereas OGT gene expression is promoted by the transcriptional activity of Nrf2 (Li et al., 2017).
Whether OGT function is affected by K63-linked protein ubiquitination, a well-established protein modification for signal transduction, has not been determined yet. In sum, similar to diverse functions of OGT through the targeting of multiple downstream targets, upstream mechanisms that modulate OGT function are also diversified.
Recent studies have identified several intracellular amyloid signaling complexes, including the necrosome (Li et al., 2012), ASC inflammasome (Lu et al., 2014), and MAVS signalosome (Cai et al., 2014), which initiate distinct important innate immune signaling.
One common feature of these high-molecular-weight complexes is well-characterized protein-protein binding patterns depending on unique domains on each protein component. The RHIM region of RIPK3, one of the best-studied protein-binding motifs, has been shown to be required for the formation of the necrosome and execution of necroptosis.
Depending on which initial sensors receive extracellular or intracellular signals, RIPK3 can directly bind to TRIF (Kaiser et al., 2013), RIPK1 (Li et al., 2012, Mompeán et al., 2018), or ZBP1 (Z-DNA binding protein 1, also known as DAI) (Lin et al., 2016, Newton et al., 2016) through the RHIM motif to activate necroptosis.
Recent studies suggest that, in addition to playing a central role in necroptosis, RIPK3 also contributes to activation of the immune response in cell-death-dependent and -independent manners (Alvarez-Diaz et al., 2016, Moriwaki et al., 2017, Najjar et al., 2016). The RIPK3 RHIM motif is critical for both necroptosis and the inflammatory response both in vitro and in vivo.
Our study identified RIPK3 T467, which is located in proximity to the RHIM region core amino acids VQVG, as an O-GlcNAcylation site. Loss of this O-GlcNAcylation promoted RHIM-mediated RIPK3-RIPK1-hetero- and RIPK3-RIPK3-homo-interaction, RIPK3 activation, downstream inflammatory and necroptosis signaling, and cytokine storm in experimental sepsis.
These findings support an essential role of RHIM-mediated assembly of the necrosome signaling complex in promoting immune activation and tissue damage in inflammatory diseases such as sepsis.
Previous studies have reported that activated RIPK3 in macrophages deficient in apoptosis-associated molecules (caspase-8 or inhibitors of apoptosis) could activate inflammasomes in response to LPS alone (Galluzzi et al., 2017, Weinlich et al., 2017). Inflammasome activation leads to caspase-1 activation and release of proinflammatory cytokines IL-1β and IL-18, among other substrates (Davis et al., 2011).
Both canonical and noncanonical inflammasomes have been indicated to play important roles promoting septic inflammation. We observed that upon LPS stimulation, loss of OGT-mediated O-GlcNAcylation caused RIPK3 phosphorylation and activation, indicating that OGT deficiency might increase RIPK3-dependent IL-1β production.
Indeed, we observed increased IL-1β concentration in septic Ogtfl/flxLyz2-cre mice, which could be reversed by Ripk3 deletion. Because IL-1β is a prototypical pyrogen to drive cytokine storm and tissue damage in sepsis, excessive IL-1β could contribute to elevated mortality in septic OgtΔmye mice.
In sum, our results provide a mechanistical link between OGT-mediated glucose metabolism and key immune signaling in the innate immune system and expand our current understanding of metabolic regulation of the immune function and inflammation-associated diseases. Targeting RIPK3 O-GlcNAcylation presents a potential therapeutic strategy for combating multiple inflammatory diseases.
More information: Yunfan Yang et al. OGT suppresses S6K1-mediated macrophage inflammation and metabolic disturbance, Proceedings of the National Academy of Sciences (2020). DOI: 10.1073/pnas.1916121117
