Nonalcoholic fatty liver disease (NAFLD) is associated with hepatic mitochondrial dysfunction characterized by reduced ATP synthesis. We applied the 2H2O-metabolic labeling approach to test the hypothesis that the reduced stability of oxidative phosphorylation proteins contributes to mitochondrial dysfunction in a diet-induced mouse model of NAFLD. A high fat diet containing cholesterol (a so-called Western diet (WD)) led to hepatic oxidative stress, steatosis, inflammation and mild fibrosis, all markers of NAFLD, in low density cholesterol (LDL) receptor deficient (LDLR-/-) mice. In addition, compared to controls (LDLR-/- mice on normal diet), livers from NAFLD mice had reduced citrate synthase activity and ATP content, suggesting mitochondrial impairment. Proteome dynamics study revealed that mitochondrial defects are associated with reduced average half-lives of mitochondrial proteins in NAFLD mice (5.41±0.46 vs. 5.15±0.49 day, P<0.05). In particular, the WD reduced stability of oxidative phosphorylation subunits, including cytochrome b-c1 complex subunit 1 (5.9 ± 0.1 vs 3.4 ± 0.8 day), ATP synthase subunit α (6.3±0.4 vs. 5.5±0.4 day) and ATP synthase F(0) complex subunit B1 of complex V (8.5±0.6 vs. 6.5±0.2 day) (P<0.05). These changes were associated with impaired complex III and F0F1-ATP synthase activities. Markers of mitophagy were increased, but markers of proteasomal degradation activity were reduced in NAFLD mice liver, suggesting that ATP deficiency due to reduced stability of oxidative phosphorylation complex subunits contributed to inhibition of ubiquitin-proteasome and activation of mitophagy. In conclusion, the 2H2O-metabolic labeling approach shows that increased degradation of hepatic oxidative phosphorylation subunits contributed to mitochondrial impairment in NAFLD mice.

Context Hyperglycemia plays a key role in the pathogenesis of cardiovascular complications of diabetes. T2DM is associated with high density lipoprotein (HDL) dysfunction and increased degradation of ApoAI. The mechanism(s) of these changes are largely unknown. Objective To study the role of hyperglycemia-induced glycation on ApoAI kinetics and stability in patients with diet-controlled T2DM. Design ²H₂O-metabolic labeling approach was used to study ApoAI turnover in patients with diet-controlled T2DM (N= 9 (5F), 59.3±8.5 yr.) and matched healthy controls (N= 8(4F), 50.7±11.6 yr.). The effect of Amadori glycation on in vivo ApoAI stability and HDL’s anti-oxidant and cholesterol efflux properties were assessed using proteomics approach and in vitro assays. Results T2DM patients had increased turnover of ApoAI, and impaired cholesterol efflux and anti-oxidant properties of HDL. HbA_1c was negatively correlated with the half-live of ApoAI, and cholesterol efflux function of HDL. Proteomic analysis identified several non-enzymatic early (Amadori) glycation of ApoAI at lysine sites. The kinetic analysis of glycated and native ApoAI peptides in T2DM patients revealed that glycation resulted in 3-fold shorter ApoAI half-life. Conclusions The ²H₂O method allows the detection of early in vivo impairments in HDL metabolism and function that are related to hyperglycemia-induced glycation of ApoAI in T2DM.

Crohn’s disease and ulcerative colitis are chronic and progressive inflammatory bowel diseases (IBDs) that are attributed to dysregulated interactions between the gut microbiome and the intestinal mucosa-associated immune system. There are limited studies investigating the role of either IL-1α or IL-1β in mouse models of colitis, and no clinical trials blocking either IL-1 have yet to be performed. In the present study, we show that neutralization of IL-1α by a specific monoclonal antibody against murine IL-1α was highly effective in reducing inflammation and damage in SAMP mice, mice that spontaneously develop a Crohn’s-like ileitis. Anti-mouse IL-1α significantly ameliorated the established, chronic ileitis and also protected mice from developing acute DSS-induced colitis. Both were associated with taxonomic divergence of the fecal gut microbiome, which was treatment-specific and not dependent on inflammation. Anti–IL-1α administration led to a decreased ratio of Proteobacteria to Bacteroidetes, decreased presence of Helicobacter species, and elevated representation of Mucispirillum schaedleri and Lactobacillus salivarius. Such modification in flora was functionally linked to the antiinflammatory effects of IL-1α neutralization, as blockade of IL-1α was not effective in germfree SAMP mice. Furthermore, preemptive dexamethasone treatment of DSS-challenged SAMP mice led to changes in flora composition without preventing the development of colitis. Thus, neutralization of IL-1α changes specific bacterial species of the intestinal microbiome, which is linked to its antiinflammatory effects. These functional findings may be of significant value for patients with IBD, who may benefit from targeted IL-1α–based therapies. Significance Here we demonstrate that IL-1α neutralization reduces the severity of disease in a mouse model of Crohn’s disease. Unexpectedly, IL-1α neutralization exerts its antiinflammatory effects by modulating the gut microbial ecosystem, and corrected the mucosal dysbiosis. Although studies report that the gut microbiome affects cytokine production, few have shown that specific neutralization of a pivotal cytokine such as IL-1α alters the composition and function of the gut microbiome. The study demonstrates a predictive relationship between IL-1α neutralization and abundance of specific bacterial species. The findings have translational significance, since IL-1α treatment prevented DSS-induced mucosal dysbiosis and preserved immunological homeostasis. These preclinical findings support the therapeutic potential of IL-1α neutralization in patients with IBD.

Gasdermin D (GSDMD)-mediated pyroptotic cell death drives inflammatory cytokine release and downstream immune responses upon inflammasome activation, which play important roles in host defense and inflammatory disorders. Upon activation by proteases, the GSDMD N-terminal domain (NTD) undergoes oligomerization and membrane translocation in the presence of lipids to assemble pores. Despite intensive studies, the molecular events underlying the transition of GSDMD from an autoinhibited soluble form to an oligomeric pore form inserted into the membrane remain incompletely understood. Previous work characterized S -palmitoylation for gasdermins from bacteria, fungi, invertebrates, as well as mammalian gasdermin E (GSDME). Here, we report that a conserved residue Cys191 in human GSDMD was S -palmitoylated, which promoted GSDMD-mediated pyroptosis and cytokine release. Mutation of Cys191 or treatment with palmitoyltransferase inhibitors cyano-myracrylamide (CMA) or 2-bromopalmitate (2BP) suppressed GSDMD palmitoylation, its localization to the membrane and dampened pyroptosis or IL-1β secretion. Furthermore, Gsdmd -dependent inflammatory responses were alleviated by inhibition of palmitoylation in vivo. By contrast, coexpression of GSDMD with palmitoyltransferases enhanced pyroptotic cell death, while introduction of exogenous palmitoylation sequences fully restored pyroptotic activities to the C191A mutant, suggesting that palmitoylation-mediated membrane localization may be distinct from other molecular events such as GSDMD conformational change during pore assembly. Collectively, our study suggests that S -palmitoylation may be a shared regulatory mechanism for GSDMD and other gasdermins, which points to potential avenues for therapeutically targeting S -palmitoylation of gasdermins in inflammatory disorders.