GSK3ß and UCHL3 govern RIPK4 homeostasis via deubiquitination to enhance tumor metastasis in ovarian cancer.

Receptor-interacting protein kinase 4 (RIPK4) is increasingly recognized as a pivotal player in ovarian cancer, promoting tumorigenesis and disease progression. Despite its significance, the posttranslational modifications dictating RIPK4 stability in ovarian cancer remain largely uncharted. In this study, we first established that RIPK4 levels are markedly higher in metastatic than in primary ovarian cancer tissues through single-cell sequencing. Subsequently, we identified UCHL3 as a key deubiquitinase that regulates RIPK4. We elucidate the mechanism that UCHL3 interacts with and deubiquitinates RIPK4 at the K469 site, removing the K48-linked ubiquitin chain and thus enhancing RIPK4 stabilization. Intriguingly, inhibition of UCHL3 activity using TCID leads to increased RIPK4 ubiquitination and degradation. Furthermore, we discovered that GSK3ß-mediated phosphorylation of RIPK4 at Ser420 enhances its interaction with UCHL3, facilitating further deubiquitination and stabilization. Functionally, RIPK4 was found to drive the proliferation and metastasis of ovarian cancer in a UCHL3-dependent manner both in vitro and in vivo. Importantly, positive correlations between RIPK4 and UCHL3 protein expression levels were observed, with both serving as indicators of poor prognosis in ovarian cancer patients. Overall, this study uncovers a novel pathway wherein GSK3ß-induced phosphorylation of RIPK4 strengthens its interaction with UCHL3, leading to increased deubiquitination and stabilization of RIPK4, thereby promoting ovarian cancer metastasis. These findings offer new insights into the molecular underpinnings of ovarian cancer and highlight potential therapeutic targets for enhancing antitumor efficacy.

Serum Lipocalin-2 Levels Are Increased and Independently Associated With Early-Stage Renal Damage and Carotid Atherosclerotic Plaque in Patients With T2DM.

Objectives: Diabetic nephropathy (DN), one of the major complications of diabetes mellitus, is the major cause of end-stage renal failure that finally increases the risk of cardiovascular disease and mortality. The aim of this study is to explore the relationship between serum lipocalin-2 (LCN-2) levels and DN and carotid atherosclerotic plaque (CAP) in patients with type 2 diabetes mellitus (T2DM). Methods: We have performed a prospective study of 749 T2DM patients with or without DN. Blood samples were collected and used to test serum LCN-2 levels, renal function, as well as biochemical parameters. CAP in these subjects was determined by ultrasonography. Results: In these 749 subjects with T2DM, an increased morbidity of CAP was observed in T2DM patients with DN as compared with those without this complication (P < 0.05). Interestingly, serum LCN-2 levels were significantly increased in T2DM patients with DN or CAP compared with T2DM alone [97.71 (71.49-130.13) vs. 77.29 (58.83-115.05) ng/ml, P < 0.001]. In addition, serum LCN-2 levels in T2DM patients with DN and CAP were significantly higher than that of T2DM patients with DN or CAP [131.37 (101.43-182.04) vs. 97.71(71.49-130.13) ng/ml, P < 0.001]. Furthermore, serum LCN-2 levels were positively correlated with hemoglobin A1c, systolic blood pressure, hypertension, CAP, and DN, as well as renal function factors including uric acid, creatinine, the estimated glomerular filtration rate, and urinary albumin-to-creatinine ratio, respectively (P < 0.05), but negatively correlated with HDL-c (P < 0.05). The multinomial logistic regression analysis showed that serum LCN-2 was independently associated with DN and CAP in patients with T2DM after the adjustment for risk factors (P < 0.001). Conclusions: Early-stage renal damage is a risk factor associated with the incidence of CAP in patients with T2DM. Serum LCN-2 is significantly increased and associated with early-stage renal damage and the incidence of CAP in patients with T2DM.

Serum FGF21 Levels Predict the MACE in Patients With Myocardial Infarction After Coronary Artery Bypass Graft Surgery.

Objectives: Prognosis evaluation in myocardial infarction (MI) patients with major adverse clinical events (MACE) who have undergone coronary artery bypass graft (CABG) is greatly important to identify high-risk patients. Elevated metabolic hormone fibroblast growth factor 21 (FGF21) is associated with the risk of MI. The aim of this study is to assess the relationship between FGF21 and the incidence of MACE in patients with MI after CABG surgery. Methods: Patients with three-vessel disease who were scheduled for first-time isolated CABG were enrolled in this project and underwent to evaluate the incidence of MACE during 48 h after CABG surgery, as well as to collect serum samples for FGF21 levels in both preoperative- and postoperative-CABG (pre-CABG and post-CABG). Results: A total of 265 patients with MI undergoing CABG were enrolled in this study, 21 patients experienced MACE during the 48 h after CAGB surgery. Serum FGF21 levels of patients with MACE at post-CABG were significantly higher than that in patients without MACE [553.7 (433.6) vs. 291.7 (334.4), p < 0.001]. Furthermore, among 81 individuals of these 265 patients, a lower level of FGF21 in preoperative-CABG (pre-CABG) and a higher level of FGF21 at postoperative-CABG (post-CABG) were observed in MI patients with MACE as compared to those without MACE respectively [ (275.0 (260.4) vs. 410.3 (420.7), p = 0.049; 550.7 (519.9) vs. 370.6 (441.2), p = 0.031]. In addition, serum FGF21 levels of MI patients with MACE at post-CABG were significantly increased compared with the baseline levels in pre-CABG [550.7 (519.9) vs.275.0 (260.4) p < 0.001]. However, these profiles were not observed in patients without MACE [410.3 (420.7) vs. 370.6 (441.2), p=0.2137]. Logistic regression analysis demonstrated that both serum FGF21 and CK-MB levels at post-CABG were independently associated with the incidence of MACE in patients with MI after CABG surgery. Finally, ROC analysis for FGF21 levels of 265 MI patients at post-CABG identified 455.4 pg/ml as an optimal cut-off value to predict MACE, with a sensitivity and specificity of 91.7 and 68.4% respectively. Conclusion: Serum FGF21 levels at post-CABG are independently associated with the incidence of MACE in patients with MI who have undergone CABG. Measurement of FGF21 may help distinguish patients with MI at a high risk of MACE after CABG surgery.

MOTS-c interacts synergistically with exercise intervention to regulate PGC-1α expression, attenuate insulin resistance and enhance glucose metabolism in mice via AMPK signaling pathway.

Mitochondrial-derived peptide (MOTS-c) has gained increasing attention as a promising therapeutic or prevention strategy for obesity and diabetes mellitus. MOTS-c targets the folate cycle, leading to an accumulation of 5-aminomidazole-4-carboxamide ribonucleotide (AICAR) as well as AMPK activation. AMPK is a well-known upstream regulator of the proliferation-activated receptor co-activator 1 (PGC-1α), which can improve mitochondrial biogenesis via co-transcriptional modifications. We hypothesized that AMPK can induce the expression of MOTS-c through PGC-1α. Our study aimed to explore whether MOTS-c and/or exercise can regulate MOTS-c expression, attenuate insulin resistance and enhance glucose metabolism both in vitro and in vivo. It was found that C2C12 myotubes exposed to Compound C (an AMPK inhibitor) had deceases in the protein and mRNA expressions of PGC-1α and MOTS-c. PGC-1α knockdown downregulated the protein and mRNA expressions of MOTS-c in C2C12 myotubes, whereas both PGC-1α overexpression and recombinant MOTS-c supplementation upregulated the protein and mRNA expressions of MOTS-c in C2C12 myotubes. Furthermore, the skeletal muscle and plasma levels of MOTS-c were markedly reduced in high-fat diet-induced obese mice. Treadmill training remarkably upregulated the protein levels of MOTS-c, PGC-1α and GLUT4, along with the phosphorylation levels of AMPK and ACC. Altogether, these results indicate that AMPK/PGC-1α pathway can mediate the secretion and/or production of MOTS-c in skeletal muscle, implying the possible roles of exercise intervention and recombinant MOTS-c in treating obesity and diabetes mellitus.

Adiponectin treatment improves insulin resistance in mice by regulating the expression of the mitochondrial-derived peptide MOTS-c and its response to exercise via APPL1-SIRT1-PGC-1α.

AIMS/HYPOTHESIS: Adiponectin stimulates mitochondrial biogenesis through peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), a major regulator of mitochondrial biogenesis. MOTS-c (mitochondrial open reading frame of the 12S rRNA) is a biologically active mitochondrial-derived peptide encoded by mitochondrial DNA. It influences the mechanisms of obesity and diabetes. We hypothesised that the adiponectin pathway may regulate the production and/or secretion of MOTS-c in skeletal muscle. We aimed to determine whether exercise and adiponectin affect MOTS-c to improve insulin resistance in mice. METHODS: To investigate this hypothesis, we used wild-type C57BL/6 mice subjected to high-fat diet, an exercise regimen, and i.p. injection of recombinant mouse adiponectin (Acrp30) or MOTS-c, and adiponectin knockout (Adipoq-/-) mice (C57BL/6 background) subjected to i.p. injection of Acrp30. C2C12 myotubes were also treated with sirtuin 1 (SIRT1) inhibitor, PGC-1α inhibitor, SIRT1 activator, plasmid-expressed active APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper), pcDNA-SIRT1, or siRNA against APPL1, SIRT1 or PGC-1α. RESULTS: In Adipoq-/- mice, MOTS-c levels in the plasma and skeletal muscle were downregulated. In C2C12 myotubes, adiponectin increased the mRNA expression of MOTS-c. APPL1 protein level following adiponectin treatment positively correlated with MOTS-c protein and mRNA levels in C2C12 myotubes. SIRT1 overexpression increased the adiponectin-induced mRNA and protein expression of MOTS-c, SIRT1 and PGC-1α. Pharmacologic and genetic inhibition of PGC-1α suppressed the increases in MOTS-c mRNA and protein levels induced by SIRT1 overexpression. In mice, plasma and skeletal muscle MOTS-c levels were significantly downregulated following high-fat-diet. Exercise and i.p. Acrp30 or MOTS-c increased MOTS-c levels and adiponectin mRNA and protein expression in the plasma and skeletal muscle. CONCLUSIONS/INTERPRETATION: Our findings showed that the APPL1-SIRT1-PGC-1α pathway regulates the production and/or secretion of skeletal muscle MOTS-c by mediating adiponectin signalling. Our study provides an insight into the cellular and molecular pathways underlying the pathogenesis of diabetes and shows that MOTS-c is a potential novel therapeutic target in the treatment of diabetes. Graphical abstract.