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New research uncovers how a natural polyamine, spermidine, modifies RIPK1 to block inflammation and metabolic damage, opening doors to innovative diabetes treatments. Image Credit: Ti_A / Shutterstock In a recent study published in the journal Nature Cell Biology , researchers investigated how N-acetyltransferase (NAT)-mediated post-translational modification, acetylhypusination, regulates insulin sensitivity and necroptosis. Type 2 diabetes (T2D) is a significant global health concern, with over 537 million adults affected.
Current T2D management approaches mainly focus on regulating hyperglycemia, which is believed to be implicated in progressive tissue/organ damage observed in the end stages of T2D. Nevertheless, the mechanisms underlying T2D onset and progression are poorly understood. Role in Longevity: Spermidine is recognized as a longevity agent due to its ability to induce autophagy, reduce inflammation, and regulate lipid metabolism, contributing to extended lifespan in model organisms and improved cellular health in humans.
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, like aliphatic amines and some drugs. Recent studies indicate that NAT2 acetylates endogenous aliphatic amines, such as spermidine and putrescine.
Spermidine is a natural polyamine found in cells whose post-translational acetylhypusination regulates key proteins like receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Aging-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 findings In the present 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 wildtype (WT) mouse embryonic fibroblasts (MEFs).
Endogenous spermidine levels in WT MEFs were ~600 μM but were significantly lower in Nat1 KO MEFs. Further, Nat1 KO MEFs had lower levels of acetylated forms than WT MEFs and had a higher sensitivity to necroptosis and receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-dependent apoptosis (RDA). However, treatment with spermidine resulted in a dose-dependent reduction in RIPK1 activation in WT and Nat1 KO MEFs.
By 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 proteins modified by spermidine, including RIPK1 and eIF5A, and validated these modifications by mass spectrometry.
Further, biotin-tagged hypusinated proteins were pulled down using streptavidin probes, and trypsin-digested peptides were quantified. Notably, RIPK1 showed a higher enrichment than eIF5A, suggesting a novel role for acetylhypusination in modulating RIPK1's 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, within 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 ninefold reduced in Nat1 KO MEFs relative to WT MEFs. Dietary Sources: Rich dietary sources of spermidine include aged cheese, mushrooms, soy products, and wheat germ, making the Mediterranean diet a particularly high source of this polyamine.
Further, conditional KO-ready mice were generated to investigate whether spermidine reductions contribute to insulin resistance in Nat1-deficient mice. The researchers observed lower levels of ac-hyp-K140 in RIPK1 in the pancreases of mice with tamoxifen-induced Nat1 deletion; spermidine levels in their pancreases were also reduced relative to WT mice. Besides, adipocyte hypertrophy (which is associated with insulin resistance and obesity) was observed after Nat1 deletion.
However, this was not observed in mice with genetically inactivated RIPK1, highlighting RIPK1's role in mediating these metabolic defects. Next, the researchers studied the vascular pathology induced by the endothelium-specific Nat1 loss. Endothelial loss of Nat1 in mice compromised blood vascular integrity in the pancreas.
Pancreases also showed robust inflammation. Interestingly, these effects were suppressed by RIPK1 inactivation, suggesting its central role in mediating vascular damage. Moreover, the team observed kidney vascular leakage in mice with Nat1 deletion; likewise, this vascular leakage was suppressed by RIPK1 inactivation.
Finally, the team estimated the levels of polyamines in vascular tissue specimens from T2D and non-T2D patients. Spermidine levels were significantly reduced in vascular tissues from T2D patients compared to those without T2D. Further, patients with diabetic nephropathy showed RIPK1 activation in kidney biopsy specimens; however, RIPK1 activation was not observed in non-diabetic nephropathy patients.
Conclusions Taken together, the results suggest a functional role of RIPK1-mediated inflammation and apoptosis in vascular pathology to promote late-stage diabetic tissue damage. Microvascular leakage may promote RIPK1-dependent inflammation, which, in turn, induces insulin resistance and obesity. As RIPK1 activation induces several pro-inflammatory cytokines, its inhibition could provide a promising therapeutic strategy to mitigate both metabolic and vascular complications in T2D.
Zhang T, Fu W, Zhang H, et al. Spermidine mediates acetylhypusination of RIPK1 to suppress diabetes onset and progression. Nature Cell Biology , 2024, DOI: 10.
1038/s41556-024-01540-6, https://www.nature.com/articles/s41556-024-01540-6.
