ITCH E3 ubiquitin ligase downregulation compromises hepatic degradation of branched-chain amino acids.

OBJECTIVE: Metabolic syndrome, obesity, and steatosis are characterized by a range of dysregulations including defects in ubiquitin ligase tagging proteins for degradation. The identification of novel hepatic genes associated with fatty liver disease and metabolic dysregulation may be relevant to unravelling new mechanisms involved in liver disease progression METHODS: Through integrative analysis of liver transcriptomic and metabolomic obtained from obese subjects with steatosis, we identified itchy E ubiquitin protein ligase (ITCH) as a gene downregulated in human hepatic tissue in relation to steatosis grade. Wild-type or ITCH knockout mouse models of non-alcoholic fatty liver disease (NAFLD) and obesity-related hepatocellular carcinoma were analyzed to dissect the causal role of ITCH in steatosis RESULTS: We show that ITCH regulation of branched-chain amino acids (BCAAs) degradation enzymes is impaired in obese women with grade 3 compared with grade 0 steatosis, and that ITCH acts as a gatekeeper whose loss results in elevation of circulating BCAAs associated with hepatic steatosis. When ITCH expression was specifically restored in the liver of ITCH knockout mice, ACADSB mRNA and protein are restored, and BCAA levels are normalized both in liver and plasma CONCLUSIONS: Our data support a novel functional role for ITCH in the hepatic regulation of BCAA metabolism and suggest that targeting ITCH in a liver-specific manner might help delay the progression of metabolic hepatic diseases and insulin resistance.

Metabolic characteristics in patients with COVID-19 and no-COVID-19 interstitial pneumonia with mild-to-moderate symptoms and similar radiological severity.

BACKGROUND AND AIMS: It is known that the highest COVID-19 mortality rates are among patients who develop severe COVID-19 pneumonia. However, despite the high sensitivity of chest CT scans for diagnosing COVID-19 in a screening population, the appearance of a chest CT is thought to have low diagnostic specificity. The aim of this retrospective case-control study is based on evaluation of clinical and radiological characteristics in patients with COVID-19 (n = 41) and no-COVID-19 interstitial pneumonia (n = 48) with mild-to-moderate symptoms. METHODS AND RESULTS: To this purpose we compared radiological, clinical, biochemical, inflammatory, and metabolic characteristics, as well as clinical outcomes, between the two groups. Notably, we found similar radiological severity of pneumonia, which we quantified using a disease score based on a high-resolution computed tomography scan (COVID-19 = 18.6 ± 14.5 vs n-COVID-19 = 23.2 ± 15.2, p = 0.289), and comparable biochemical and inflammatory characteristics. However, among patients without diabetes, we observed that COVID-19 patients had significantly higher levels of HbA1c than n-COVID-19 patients (COVID-19 = 41.5 ± 2.6 vs n-COVID-19 = 38.4 ± 5.1, p = 0.012). After adjusting for age, sex, and BMI, we found that HbA1c levels were significantly associated with the risk of COVID-19 pneumonia (odds ratio = 1.234 [95%CI = 1.051-1.449], p = 0.010). CONCLUSIONS: In this retrospective case-control study, we found similar radiological and clinical characteristics in patients with COVID-19 and n-COVID-19 pneumonia with mild-to-moderate symptoms. However, among patients without diabetes HbA1c levels were higher in COVID-19 patients than in no-COVID-19 individuals. Future studies should assess whether reducing transient hyperglycemia in individuals without overt diabetes may lower the risk of SARS-CoV-2 infection.

Iron status influences non-alcoholic fatty liver disease in obesity through the gut microbiome.

BACKGROUND: The gut microbiome and iron status are known to play a role in the pathophysiology of non-alcoholic fatty liver disease (NAFLD), although their complex interaction remains unclear. RESULTS: Here, we applied an integrative systems medicine approach (faecal metagenomics, plasma and urine metabolomics, hepatic transcriptomics) in 2 well-characterised human cohorts of subjects with obesity (discovery n = 49 and validation n = 628) and an independent cohort formed by both individuals with and without obesity (n = 130), combined with in vitro and animal models. Serum ferritin levels, as a markers of liver iron stores, were positively associated with liver fat accumulation in parallel with lower gut microbial gene richness, composition and functionality. Specifically, ferritin had strong negative associations with the Pasteurellaceae, Leuconostocaceae and Micrococcaea families. It also had consistent negative associations with several Veillonella, Bifidobacterium and Lactobacillus species, but positive associations with Bacteroides and Prevotella spp. Notably, the ferritin-associated bacterial families had a strong correlation with iron-related liver genes. In addition, several bacterial functions related to iron metabolism (transport, chelation, heme and siderophore biosynthesis) and NAFLD (fatty acid and glutathione biosynthesis) were also associated with the host serum ferritin levels. This iron-related microbiome signature was linked to a transcriptomic and metabolomic signature associated to the degree of liver fat accumulation through hepatic glucose metabolism. In particular, we found a consistent association among serum ferritin, Pasteurellaceae and Micrococcacea families, bacterial functions involved in histidine transport, the host circulating histidine levels and the liver expression of GYS2 and SEC24B. Serum ferritin was also related to bacterial glycine transporters, the host glycine serum levels and the liver expression of glycine transporters. The transcriptomic findings were replicated in human primary hepatocytes, where iron supplementation also led to triglycerides accumulation and induced the expression of lipid and iron metabolism genes in synergy with palmitic acid. We further explored the direct impact of the microbiome on iron metabolism and liver fact accumulation through transplantation of faecal microbiota into recipient's mice. In line with the results in humans, transplantation from 'high ferritin donors' resulted in alterations in several genes related to iron metabolism and fatty acid accumulation in recipient's mice. CONCLUSIONS: Altogether, a significant interplay among the gut microbiome, iron status and liver fat accumulation is revealed, with potential significance for target therapies. Video abstract.

IL-6 Levels Influence 3-Month All-Cause Mortality in Frail Hospitalized Older Patients.

The multidimensional prognostic index (MPI) is a sensitive and specific prognosis estimation tool that accurately predicts all-cause mortality in frail older patients. It has been validated to assess the risk of 1-month to 2-year mortality in frail older patients during hospitalization and after hospital discharge. However, whether the MPI is a valid prognostic tool for follow-up periods of different lengths remains to be validated. To this end, we followed up 80 hospitalized patients (female=37, male 43) at least 75 years of age (mean age=82.6±4.4, range=75-94 years) to assess the 3-month all-cause mortality (mean follow-up=61.0 ± 31.7 months [range 4-90 days]). Accordingly, patients were subdivided into low (MPI-1, score 0-0.33), moderate (MPI-2, score 0.34-0.66) and high (MPI-3, score 0.67-1) mortality risk classes. Moreover, baseline biochemical, inflammatory and metabolic parameters, as well as anamnestic and clinical characteristics, were obtained. Although the MPI-3 score was significantly associated with 3-month all-cause mortality in univariate analysis (HR=5.79, 95%CI=1.77-18.92, p=0.004), a multivariate model indicated that only low albumin (HR=0.33, 95%CI=0.16-0.68, p=0.003) and high IL6 (HR=1.01, 95%CI=1.00-1.02, p=0.010) levels were significantly associated with 3-month all-cause mortality. In conclusion, we suggest that measurement of IL6 as well as albumin, rather than the MPI score, may help in providing tailored therapeutic interventions to decrease short term mortality in older hospitalized individuals.

Chronic Kidney Disease Is Linked to Carotid Nodular Calcification, An Unstable Plaque Not Correlated to Inflammation.

The incidence and the different type of carotid calcifications, nodular and non-nodular, and their role in the acute cerebrovascular disease has not yet been defined. Various studies have correlated the presence of specific risk factors, in particular the chronic kidney disease, with the presence of calcification, but not with the type of calcification. Since it is likely that carotid nodular calcifications rather than those with non-nodular aspect may represent a plaque at high risk of rupture, the purpose of our study was to evaluate the role of nodular calcification in the pathogenesis of cerebrovascular syndromes and their possible correlation with specific risk factors. A total of 168 carotid plaques from symptomatic and asymptomatic patients submitted to endarterectomy, whom complete clinical and laboratory assessment of major cardiovascular risk factors was available, were studied. In 21 endarterectomies (5 from symptomatic and 16 from asymptomatic patients) an eruptive calcified nodule, consisting of calcified plates associated to a small amount of fibrous tissue without extracellular lipids and inflammatory cells, was found protruding into the lumen. Nodular calcifications were significantly observed in patients affected by chronic kidney disease (with GFR<60 ml / min / 1.73 m2), with a normal lipidic and glycemic profile. On the contrary, non-nodular calcification, mainly correlated to diabetes, were stable lesions. Results of our study suggest that the mechanisms and the clinical significance of carotid atherosclerotic calcification may be different. The nodular calcification could represent a type of unstable plaque, significantly related to chronic kidney disease, without inflammation, morphologically different from the classical vulnerable plaques.

Soluble ST2 is a biomarker for cardiovascular mortality related to abnormal glucose metabolism in high-risk subjects.

AIMS: Inflammation plays a role in the development and progression of type 2 diabetes macroangiopathy. Interleukin 33 (IL-33) drives production of Th2-associated cytokines. The soluble form of suppression of tumorigenicity 2 (sST2) acting as a decoy receptor blocks IL-33 and tones down Th2 inflammatory response. We investigated the role of sST2 as a predictor of CV and all-cause mortality in a cohort of patients affected by established atherosclerotic disease. METHODS: 399 patients with atherosclerotic disease from the Tor Vergata Atherosclerosis Registry performed follow-up every year by phone interview. The primary endpoint was cardiovascular death and the secondary endpoint was death for any other disease. RESULTS: sST2 plasma levels were significantly increased from normal glucose-tolerant patients to patients with history of type 2 diabetes (p < 0.00001). Levels of sST2 were significantly correlated with fasting plasma glucose (R = 0.16, p = 0.002), HbA1c (R = 0.17, p = 0.002), and HOMA (R = 0.16, p = 0.004). Dividing patients in tertiles of sST2 levels, those belonging to the highest tertile showed an increased rate of all-cause and cardiovascular mortality, (all-cause mortality p = 0.045 and CVD mortality p = 0.02). A multivariate Cox analysis revealed that sST2 increased the risk in cardiovascular mortality per SD by hazard ratio 1.050 (95% CI 1.006-1.097, p = 0.025) after adjustment for age and hs-CRP while it did not significantly change the risk for all-cause mortality. CONCLUSIONS: High circulating level of sST2 is associated to increased CVD mortality and markers of metabolic dysfunction in subjects with atherosclerotic disease.

Publisher Correction: Molecular phenomics and metagenomics of hepatic steatosis in non-diabetic obese women.

In the version of this article originally published, the received date was missing. It should have been listed as 2 January 2018. The error has been corrected in the HTML and PDF versions of this article.

2-hydroxycaproate predicts cardiovascular mortality in patients with atherosclerotic disease.

BACKGROUND AND AIMS: We aimed to identify novel biomarkers for cardiovascular mortality through a non-targeted metabolomics approach in patients with established atherosclerotic disease from the Tor Vergata Atherosclerosis Registry (TVAR). METHODS: We compared the serum baseline metabolome of 19 patients with atherosclerosis suffering from cardiovascular death during follow-up with the baseline serum metabolome of 20 control patients matched for age, gender, body mass index (BMI) and atherosclerotic disease status, who survived during the observation period. RESULTS: Three metabolites were significantly different in the cardiovascular mortality (CVM) group compared to controls: 2-hydroxycaproate, gluconate and sorbitol. 2-hydroxycaproate (otherwise known as alpha hydroxy caproate) was also significantly correlated with time to death. The metabolites performed better when combined together rather than singularly on the identification of CVM status. CONCLUSIONS: Our analysis led to identify few metabolites potentially amenable of translation into the clinical practice as biomarkers for specific metabolic changes in the cardiovascular system in patients with established atherosclerotic disease.

Molecular phenomics and metagenomics of hepatic steatosis in non-diabetic obese women.

Hepatic steatosis is a multifactorial condition that is often observed in obese patients and is a prelude to non-alcoholic fatty liver disease. Here, we combine shotgun sequencing of fecal metagenomes with molecular phenomics (hepatic transcriptome and plasma and urine metabolomes) in two well-characterized cohorts of morbidly obese women recruited to the FLORINASH study. We reveal molecular networks linking the gut microbiome and the host phenome to hepatic steatosis. Patients with steatosis have low microbial gene richness and increased genetic potential for the processing of dietary lipids and endotoxin biosynthesis (notably from Proteobacteria), hepatic inflammation and dysregulation of aromatic and branched-chain amino acid metabolism. We demonstrated that fecal microbiota transplants and chronic treatment with phenylacetic acid, a microbial product of aromatic amino acid metabolism, successfully trigger steatosis and branched-chain amino acid metabolism. Molecular phenomic signatures were predictive (area under the curve = 87%) and consistent with the gut microbiome having an effect on the steatosis phenome (>75% shared variation) and, therefore, actionable via microbiome-based therapies.

C-peptide: A predictor of cardiovascular mortality in subjects with established atherosclerotic disease.

AIM: Insulin resistance and type 2 diabetes are independent risk factors for cardiovascular diseases. Levels of C-peptide are increased in these patients and its role in the atherosclerosis progression was studied in vitro and in vivo over the past years. To evaluate the possible use of C-peptide as cardiovascular biomarkers, we designed an observational study in which we enrolled patients with mono- or poly-vascular atherosclerotic disease. METHODS: We recruited 431 patients with stable atherosclerosis and performed a yearly follow-up to estimate the cardiovascular and total mortality and cardiovascular events. RESULTS: We performed a mean follow-up of 56 months on 268 patients. A multivariate Cox analysis showed that C-peptide significantly increased the risk of cardiovascular mortality [Hazard Ratio: 1.29 (95% confidence interval: 1.02-1.65, p < 0.03513)] after adjustment for age, sex, diabetes treatment, estimated glomerular filtration rate and known diabetes status. Furthermore, levels of C-peptide were significantly correlated with metabolic parameters and atherogenic factors. CONCLUSION: C-peptide was associated with cardiovascular mortality independently of known diabetes status in a cohort of patients with chronic atherosclerotic disease. Future studies using C-peptide into a reclassification approach might be undertaken to consider its potential as a cardiovascular disease biomarker.

FoxO1 regulates asymmetric dimethylarginine via downregulation of dimethylaminohydrolase 1 in human endothelial cells and subjects with atherosclerosis.

BACKGROUND AND AIMS: The O subfamily of forkhead (FoxO) 1 is a pivotal element in the regulation of endothelial activation. Compartmentalization and activity of FoxO1 is regulated by post translational modifications, but the implication in endothelial dysfunction and atherosclerosis remain controversial. Our aim was to identify FoxO1 related metabolic signatures in endothelial cells. METHODS AND RESULTS: Using metabolomics in human umbilical endothelial cells (HUVECs) overexpressing the wild type FoxO1 (FoxO1-WT), the acetylation defective mutant (FoxO1-KR), the unphosphorylated nuclear localized mutant (FoxO1-ADA) and the Green Fluorescent Protein (GFP) control vector, we identify metabolic pathways differentially affected by the different FoxO1 localization and activity. Among metabolites, asymmetric dimethylarginine (ADMA) was increased in FoxO1-ADA compared with FoxO1-WT and FoxO1-KR infected cells (p < 0.01). ADMA was further investigated to identify the molecular mechanisms to explain its link to FoxO1. We found that unrestrained FoxO1 activity leads to increase of ADMA via downregulation of its degrading enzyme, dimethylaminohydrolase (DDAH) 1. In human subjects (n = 89) the FoxO1/DDAH1/ADMA pathway marks unstable atherosclerosis. CONCLUSIONS: Our results point to ADMA as a biomarker to track deregulated FoxO1 activity in vivo.

Decreased IRS2 and TIMP3 expression in monocytes from offspring of type 2 diabetic patients is correlated with insulin resistance and increased intima-media thickness.

OBJECTIVE: In humans, it is unclear if insulin resistance at the monocyte level is associated with atherosclerosis in vivo. Here we have studied first-degree relatives of patients with type 2 diabetes to investigate whether a reduction in components of the insulin signal transduction pathways, such as the insulin receptor (InsR) or InsR substrate 1 or 2 (IRS1 or IRS2), or a reduction in genetic modifiers of insulin action, such as the TIMP3/ADAM17 (tissue inhibitor of metalloproteinase 3/A disintegrin and metalloprotease domain 17) pathway, is associated with evidence of atherosclerosis. RESEARCH DESIGN AND METHODS: Insulin sensitivity was analyzed through euglycemic-hyperinsulinemic clamp, and subclinical atherosclerosis was analyzed through intimal medial thickness. Monocytes were isolated through magnetic cell sorting, and mRNA and proteins were extracted and analyzed by quantitative PCR and pathscan enzyme-linked immunosorbent assays, respectively. RESULTS: In monocyte cells from human subjects with increased risk for diabetes and atherosclerosis, we found that gene expression, protein levels, and tyrosine phosphorylation of IRS2, but not InsR or IRS1, were decreased. TIMP3 was also reduced, along with insulin resistance, resulting in increased ectodomain shedding activity of the metalloprotease ADAM17. CONCLUSIONS: Systemic insulin resistance and subclinical atherosclerosis are associated with decreased IRS2 and TIMP3 expression in circulating monocytes.