New research reveals how a natural polyamine, spermidine, modifies RIPK1 to block inflammation and metabolic damage, opening the door to innovative treatments for diabetes.
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In a recent study published in the journal Natural cellular biologyResearchers investigated how N-acetyltransferase (NAT)-mediated post-translational modification, acetylhypusination, regulates insulin sensitivity and necroptosis.
Type 2 diabetes (T2D) is a significant global health problem, with more than 537 million adults affected. Current approaches to the management of T2D mainly focus on the regulation of hyperglycemia, which is thought to be involved in the progressive tissue/organ damage observed in the end stages of T2D. However, the mechanisms underlying the onset and progression of T2DM are poorly understood.
The gene encoding human NAT2 (hNAT2), an ortholog of murine Nat1 (mNAT1), has been reported to mediate insulin sensitivity. hNAT2 and mNAT1 serve as arylamine N-acetyltransferases in the xenobiotic metabolism of exogenous molecules, such as aliphatic amines and certain drugs. Recent studies indicate that NAT2 acetylates endogenous aliphatic amines, such as spermidine and putrescine.
Spermidine is a natural polyamine present in cells whose post-translational acetylhypusination regulates key proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Age-related reductions in spermidine levels have been reported in humans and mice, and its supplementation has been suggested to slow aging and promote health. Spermidine is involved in hypusination, a post-translational modification. Eukaryotic translation initiation factor 5A (eIF5A) is the only substrate known to be modified by hypusination.
The study and results
In the current study, researchers explored how hNAT2 and mNAT1 regulate insulin sensitivity and necroptosis. First, they quantified spermine, putrescine, and spermidine and their acetylated forms in Nat1 knockout (KO) and wild-type (WT) mouse embryonic fibroblasts (MEFs). Endogenous spermidine levels in WT MEFs were approximately 600 µM but were significantly lower in Nat1 KO MEFs.
Furthermore, Nat1 KO MEFs had lower levels of acetylated forms than WT MEFs and exhibited higher sensitivity to serine/threonine-interacting protein kinase 1 (RIPK1)-dependent necroptosis and apoptosis (RDA). with the receptors. However, spermidine treatment resulted in a dose-dependent reduction in RIPK1 activation in both WT and Nat1 KO MEFs.
In contrast, putrescine treatment did not affect necroptosis or RDA. Next, the team synthesized an alkyne-spermidine probe and treated WT MEFs and deoxyhypusine synthase (Dhps) KO MEFs with this probe. Using click chemistry, the team identified 1,895 spermidine-modified proteins, including RIPK1 and eIF5A, and validated these modifications using mass spectrometry.
Additionally, biotin-labeled hypusinated proteins were extracted using streptavidin probes and trypsin-digested peptides were quantified. Notably, RIPK1 showed higher enrichment than eIF5A, suggesting a novel role of acetylhypusination in modulating RIPK1 activity.
Next, the team used mass spectrometry to investigate potential hypusination sites in RIPK1 in Nat1 KO and WT MEFs. This identified an acetylhypusination site (K140), ac-hyp-K140, in the kinase domain and hypusination sites in the kinase (K226) and intermediate (K550) domains. The researchers focused on the K140 site, given that ac-hyp-K140 was reduced ninefold in Nat1 KO MEFs compared to WT MEFs.
Additionally, conditional KO-ready mice were generated to determine whether spermidine reductions contribute to insulin resistance in Nat1-deficient mice. Researchers observed lower levels of ac-hyp-K140 in RIPK1 in the pancreas of mice with tamoxifen-induced Nat1 deletion; Spermidine levels in their pancreas were also reduced compared to WT mice.
Furthermore, adipocyte hypertrophy (associated with insulin resistance and obesity) was observed after Nat1 deletion. However, this was not observed in mice with genetically inactivated RIPK1, highlighting the role of RIPK1 in mediating these metabolic defects. Next, the researchers studied vascular pathology induced by endothelium-specific loss of Nat1.
Endothelial loss of Nat1 in mice compromised pancreatic blood vascular integrity. The pancreases also showed robust inflammation. Interestingly, these effects were abolished by RIPK1 knockdown, suggesting its central role in mediating vascular injury. Additionally, the team observed renal vascular leakage in mice with Nat1 deletion; similarly, this vascular leak was suppressed by RIPK1 inactivation.
Finally, the team estimated polyamine levels in vascular tissue samples from patients with and without T2D. Spermidine levels were significantly reduced in vascular tissues of patients with T2DM compared to those without T2DM. Additionally, patients with diabetic nephropathy showed RIPK1 activation in kidney biopsy samples; however, RIPK1 activation was not observed in patients with non-diabetic nephropathy.
Conclusions
Taken together, the results suggest a functional role of RIPK1-mediated inflammation and apoptosis in vascular pathology to promote late-stage diabetic tissue injury. Microvascular leakage can promote RIPK1-dependent inflammation, which, in turn, induces insulin resistance and obesity. As RIPK1 activation induces several pro-inflammatory cytokines, its inhibition could be a promising therapeutic strategy to alleviate the metabolic and vascular complications of T2D.
