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Type 2 diabetes (T2D) is a multifactorial condition whose greatest impact comes from its complications. Not only impaired glucose homeostasis, but also lipid alterations have a relevant role, with insulin derived mechanisms behind this milieu, i.e., glycemia and lipidemia. Thus, we hypothesized that a) distinct glucose and lipid profiles and b) sex differences, particularly in lipids patterns, may be used to identify subjects at higher risk to develop T2D.The PREVADIAB2 study evaluated metabolic alterations after 5 years in subjects without T2D when participating to PREVADIAB1. Herein, 953 subjects from the PREVADIAB2 cohort were stratified using a hierarchical clustering algorithm, informed by HOMA-IR, IGI,fISR andfIC. The resulting clusters were profiled and the lipidome of a subset (n=488) was assessed by LC/MS-QTOF.We identified four clusters, named according to their main metabolic features: Liver Sensitive (LS); Pancreas Glucose Sensitive (PGS); Insulin Deficient (ID); and Insulin Resistant (IR). These cluster metabolic patterns were similar between sexes. However, men and women had distinct parameters cut-offs and lipidomic profiles. Overall, women presented higher long chain ceramides. Nonetheless, men had higher ceramide to sphingomyelin ratio and higher lysophosphatidylcholine to phosphatidylcholine ratio. For both genders, the LS cluster had the most advantageous lipid profile, whereas the other clusters presented lipid specificities towards dysmetabolism. This work shows that clustering individuals by distinct insulin-related metabolic features and sex identifies different phenotypes with distinct lipidome profile, thus demonstrating the importance of placing diabetes in a broader context of metabolism beyond glucose.

Little is known about the impact of metabolic stimuli on brain tissue at a molecular level. The ketone body beta-hydroxybutyrate (BHB) can be a signaling molecule regulating gene transcription. Thus, we assessed lysine beta-hydroxybutyrylation (K-bhb) levels in proteins extracted from the cerebral cortex of mice undergoing a ketogenic metabolic challenge (48 h fasting). We found that fasting enhanced K-bhb in a variety of proteins including histone H3. ChIP-seq experiments showed that K9 beta-hydroxybutyrylation of H3 (H3K9-bhb) was significantly enriched by fasting on more than 8000 DNA loci. Transcriptomic analysis showed that H3K9-bhb on enhancers and promoters correlated with active gene expression. One of the most enriched functional annotations both at the epigenetic and transcriptional level was "circadian rhythms''. Indeed, we found that the diurnal oscillation of specific transcripts was modulated by fasting at distinct zeitgeber times both in the cortex and suprachiasmatic nucleus. Moreover, specific changes in locomotor activity daily features were observed during re-feeding after 48-h fasting. Thus, our results suggest that fasting remarkably impinges on the cerebral cortex transcriptional and epigenetic landscape, and BHB acts as a powerful epigenetic molecule in the brain through direct and specific histone marks remodeling in neural tissue cells.

Phthalates and bisphenol A (BPA) are plasticizers used in many industrial products that can act as endocrine disruptors and lead to metabolic diseases. During the LIFE PERSUADED project, we measured the urinary concentrations of BPA and Di(2-ethylhexyl)phthalate (DEHP) metabolites in 900 Italian women representative of the Italian female adult population (living in the north, centre, and south of Italy in both rural and urban areas). The whole cohort was exposed to DEHP and BPA with measurable levels above limit of detection in more than 99% and 95% of the samples, respectively. The exposure patterns differed for the two chemicals in the three macro-areas with the highest urinary levels for DEHP in south compared to central and northern Italy and for BPA in northern compared to central and southern Italy. BPA levels were higher in women living in urban areas, whereas no difference between areas was observed for DEHP. The estimated daily intake of BPA was 0.11 μg/kg per day, about 36-fold below the current temporary tolerable daily intake of 4 μg/kg per day established by the EFSA in 2015. The analysis of cumulative exposure showed a positive correlation between DEHP and BPA. Further, the reduction of exposure to DEHP and BPA, through specific legislative measures, is necessary to limit the harmfulness of these substances.

BACKGROUND AND AIMS Peroxisome proliferator-activated receptor (PPAR)-γ agonists decrease hepatic/visceral fat (VF) and improve necroinflammation despite subcutaneous (SC) fat weight-gain. Understanding the impact of changes in VF, VF-to-SC fat distribution (VF/SC) and adiponectin (ADPN) levels in relation to histological improvement after weight-loss or pioglitazone is relevant as novel PPAR-γ agonists are being developed for treating non-alcoholic steatohepatitis (NASH). METHODS Fifty-five patients with NASH received a -500 kcal/d hypocaloric diet and were randomized (double-blind) to pioglitazone (45 mg/d) or placebo for 6-months. Before and after treatment patients underwent a liver biopsy and measurement of hepatic/peripheral glucose fluxes, hepatic/adipose tissue-IR and, in 35 patients, hepatic and VF/SC-fat was measured by magnetic resonance spectroscopy/imaging. Data were examined by multivariable statistical analyses combined with machine-learning techniques (partial least square discriminant analysis [PLS-DA]). RESULTS Both pioglitazone (despite weight-gain) and placebo (if weight-loss) reduced steatosis but only pioglitazone ameliorated necroinflammation. Using machine-learning PLS-DA showed that the treatment differences induced by a PPAR-γ agonist vs placebo on metabolic variables and liver histology could be best explained by the increase in ADPN and a decrease in VF/SC, and to a lesser degree, improvement in oral glucose tolerance test-glucose concentrations and ALT. Decrease in steatosis and disease activity score (ballooning plus lobular inflammation) kept a close relationship with an increase in ADPN (r = -.71 and r = -.44, P

Non-alcoholic fatty liver disease (NAFLD) is a heterogeneous disorder, but the factors that determine this heterogeneity remain poorly understood. Adipose tissue dysfunction is causally linked to NAFLD since it causes intrahepatic triglyceride (IHTG) accumulation through increased hepatic lipid flow, due to insulin resistance and pro-inflammatory adipokines release. While many studies in NAFLD have looked at total adiposity (i.e. mainly subcutaneous fat, SC-AT), it is still unclear the possible impact of visceral fat (VF). Thus, we investigated how VF versus SC-AT was related to NAFLD severity in lean, overweight and obese individuals versus lean controls.Thirty-two non-diabetic NAFLD with liver biopsy (BMI 21.4-34.7 kg/mIntrahepatic triglyceride was positively associated with lipolysis, adipose tissue insulin resistance (Adipo-IR), TAG concentrations, and increased saturated/unsaturated FFA ratio. Compared to controls VF was higher in NAFLD (including lean individuals), increased with fibrosis stage and associated with insulin resistance in liver, muscle and adipose tissue, increased lipolysis and decreased adiponectin levels. Collectively, our results suggest that VF accumulation, given its location close to the liver, is one of the major risk factors for NAFLD.These findings propose VF as an early indicator of NAFLD progression independently of BMI, which may allow for evidence-based prevention and intervention strategies.

Although the effect of glucagon on glucose metabolism has been well characterized, its effect on lipid metabolism, particularly lipolysis and de novo lipogenesis, in humans in vivo has been poorly studied with controversial results. We examined the effect of short-term hyperglucagonemia on de novo lipogenesis (DNL) and endogenous glucose production (EGP) in 8 healthy normal glucose tolerant subjects (5M/3F, age=35±5, BMI= 24±1) who received a 12-hour (6PM to 6AM) glucagon infusion (6ng/kg/min) infusion with measurement of EGP (3-3H-glucose) on the following morning 6-AM. Subjects received deuterated water (D2O, 5g/kg fat free mass) at PM on the night prior to study. 8 weeks later subjects returned for a repeat study with 12-hour infusion of normal saline. DNL was quantified by measuring the incorporation of deuterated water into circulating triglycerides. Gluconeogenic rate was measured as (EGP) x (plasma C5/C2 glucose ratio) (EGP) x (plasma C5/C2 glucose ratio) . Plasma esterified and non-esterified FFA levels were measured with gas chromatography mass spectrometry (GC-MS) . Plasma glucagon increased from 57±3 to 219±21 pg/ml within one hour and remained elevated throughout the study. DNL was significantly decreased (1.64 ± 0.58% vs. 3.± 0.98%, p Disclosure X.Chen: None. F.Carli: None. S.Pezzica: None. G.Mocciaro: None. D.Ciociaro: None. A.Gastaldelli: Advisory Panel; Boehringer Ingelheim International GmbH, Novo Nordisk Global Business Services, Consultant; Boehringer Ingelheim International GmbH, Inventiva Pharma, Other Relationship; Eli Lilly and Company, Gilead Sciences, Inc., Pfizer Inc., Speaker's Bureau; Novo Nordisk. R.A.Defronzo: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Intarcia Therapeutics, Inc., Novo Nordisk, Research Support; AstraZeneca, Boehringer Ingelheim International GmbH, Merck & Co., Inc., Speaker's Bureau; AstraZeneca. D.Tripathy: None. Funding EFSD Albert Renold Travel Fellowship; FAVHR

Phthalates and bisphenol A (BPA) are plasticizers used in many industrial products that can act as endocrine disruptors. In the frame of the LIFE PERSUADED project, we measured the urinary concentrations of BPA and Di (2-ethylhexyl) phthalate (DEHP) metabolites in 900 Italian women enrolled between 2015 and 2017 and living in North, Centre and South of Italy in both rural and urban areas.The whole cohort, representative of the Italian female adult population, was exposed to DEHP and BPA with measurable levels above the limit of detection (LOD) in more than 99% and 95% of the samples, respectively. The exposure patterns differed for the two chemicals in the three macro-areas with the highest urinary levels for DEHP in South compared to Central and Northern Italy and for BPA in Northern compared to Central and Southern Italy. BPA levels were higher in women living in urban areas, whereas no difference between areas was observed for DEHP. The estimated daily intake of BPA was 0.11 μg/kg per day, about 36-fold below the current temporary Tolerable Daily Intake of 4 μg/kg per day established by the European Food Safety Authority in 2015. The analysis of cumulative exposure showed a positive correlation between DEHP and BPA.These results suggest to further limit the exposure to DEHP and BPA through specific legislative measures.

Background & AimsNonalcoholic fatty liver disease (NAFLD) is a heterogeneous disorder, but the factors that determine this heterogeneity remain poorly understood. Adipose tissue (AT) dysfunction is causally linked to NAFLD since it causes intrahepatic triglyceride (IHTG) accumulation through increased hepatic lipid flow, due to insulin resistance (IR) and pro-inflammatory adipokines release. While many studies in NAFLD have looked at total adiposity (that is mainly subcutaneous fat, SC-AT), it is still unclear the impact of visceral fat (VF). Thus, we investigated how VF vs. SC-AT were related to NAFLD in lean, overweight, and obese individuals compared to lean controls.MethodsThirty-four non-diabetic NAFLD with liver biopsy and eight lean control individuals (CT) were enrolled in this study. We measured fat distribution (VF, SC-AT and IHTG) by magnetic resonance imaging (MRI), adiponectin concentration, free fatty acids (FFAs) and triglyceride (TAG) concentration and composition by mass spectrometry (MS), lipolysis and IR by tracer infusion.ResultsIHTG was positively associated with lipolysis, adipose tissue IR, TG concentrations, and increased ratio of saturated/unsaturated fatty acids. VF was higher in NAFLD (including lean individuals) compared to controls, was increased with fibrosis stage and was associated with IR in liver, muscle and adipose tissue, increased lipolysis, and decreased adiponectin levels. Collectively, our results suggest that VF accumulation, given its location close to the liver, is one of the major risk factors for NAFLD.ConclusionsThese findings propose VF as an early indicator of NAFLD independently of BMI, which may allow for evidence-based prevention and intervention strategies.

Background & Aims The I148M Patatin-like Phospholipase Domain-containing 3 (PNPLA3), the rs641738 in the Membrane bound O-acyltransferase domain containing 7-transmembrane channel-like 4 (MBOAT7-TMC4) locus, and the E167K Transmembrane 6 Superfamily Member 2 (TM6SF2) polymorphisms represent the main predisposing factors to nonalcoholic fatty liver disease (NAFLD) development and progression. We previously generated a full knockout of MBOAT7 in HepG2 cells (MBOAT7-/-), homozygous for I148M PNPLA3. Therefore, we aimed to investigate the synergic impact of the 3 at-risk variants on liver injury and hepatocellular carcinoma (HCC) in a large cohort of NAFLD patients, and create in vitro models of genetic NAFLD by silencing TM6SF2 in both HepG2 and MBOAT7-/- cells. Methods NAFLD patients (n = 1380), of whom 121 had HCC, were stratified with a semiquantitative score ranging from 0 to 3 according to the number of PNPLA3, TM6SF2, and MBOAT7 at-risk variants. TM6SF2 was silenced in HepG2 (TM6SF2-/-) and MBOAT7-/- (MBOAT7-/-TM6SF2-/-) through Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Results In NAFLD patients, the additive weight of these mutations was associated with liver disease severity and an increased risk of developing HCC. In HepG2 cells, TM6SF2 silencing altered lipid composition and induced the accumulation of microvesicular lipid droplets (LDs), whereas the MBOAT7-/-TM6SF2-/- cells showed a mixed microvesicular/macrovesicular pattern of LDs. TM6SF2 deletion strongly affected endoplasmic reticulum and mitochondria ultrastructures, thus increasing endoplasmic reticulum/oxidative stress. The mitochondrial number was increased in both TM6SF2-/- and MBOAT7-/-TM6SF2-/- models, suggesting an unbalancing in mitochondrial dynamics, and the silencing of both MBOAT7 and TM6SF2 impaired mitochondrial activity with a shift toward anaerobic glycolysis. MBOAT7-/-TM6SF2-/- cells also showed the highest proliferation rate. Finally, the re-overexpression of MBOAT7 and/or TM6SF2 reversed the metabolic and tumorigenic features observed in the compound knockout model. Conclusions The co-presence of the 3 at-risk variants impacts the NAFLD course in both patients and experimental models, affecting LD accumulation, mitochondrial functionality, and metabolic reprogramming toward HCC., Graphical abstract

Background: Exercise may affect lipid profile which in turn is related to inflammation, although changes of ceramides, diacylglycerols-DAG and sphingomyelin-SM and their relationship with inflammatory parameters following a half-marathon have never been examined. Methods: Ceramides, DAG and SM, and markers of inflammation (soluble fractalkine-CX3CL1, vascular endothelial growth factor-VEGF, interleukin6-IL-6 and tumor necrosis factorα-TNFα) were evaluated in trained half-marathoners before, post-race (withdrawal within 20 min after the race end) and 24 h after. Results: IL-6 and CX3CL1 increased immediately after the race, returning to baseline after 24 h. Total ceramides and total DAG significantly decreased post-race. Several ceramide classes decreased after exercise, while only one of the DAG (36:3) changed significantly. Total SM and specific species did not significantly change. Conclusion: Some inflammatory parameters (IL-6 and CX3CL1) transiently increased after the race, and, being reversible, these changes might represent a physiological response to acute exercise rather than a damage-related response. The decrease of specific lipid classes, i.e., DAGs and ceramides, and the lack of their relationship with inflammatory parameters, suggest their involvement in beneficial training effects, opening promising research perspectives to identify additional mechanisms of aerobic exercise adaptation.

Aims: Non-Alcoholic Fatty Liver Disease (NAFLD) and type 2 diabetes (T2D) share pathophysiological mechanisms and possible therapeutic strategies. We evaluated the effects of 1-year treatment with pioglitazone or sulphonylureas on indirect indices of NAFLD in people with T2D and the role of insulin-resistance and glucotoxicity in determining these effects. Methods: Patients with T2D (n = 195) aged 50–75 years, poorly controlled with metformin 2 g/day, were randomly allocated to add-on pioglitazone (n = 98) or sulphonylureas (n = 97) within the TOSCA.IT trial. Plasma insulin, glucose, and liver enzymes were measured at baseline and after 1-year. Indirect indices of NAFLD (Liver Fat Equation [LFE], Hepatic Steatosis Index [HSI], and Index of NASH [ION]), and insulin resistance (HOMA-IR, Visceral Adiposity Index [VAI] and adipose tissue Insulin Resistance [ADIPO-IR]) were calculated. Results: Indices of NAFLD improved after pioglitazone, but not after sulphonylureas; differences between changes (1-year minus baseline) were respectively: -1.76 ± 3.84 vs. 0.28 ± 3.75 for LFE; -1.35 ± 2.78 vs. -0.27 ± 2.63 for HSI; -9.75 ± 43 vs. 3.24 ± 31 for ION; p < 0.05 for all. Indices of insulin resistance decreased after pioglitazone, but not after sulphonylureas: -0.95 ± 4.57 vs. 0.37 ± 3.34 for HOMA-IR, p = 0.032; -1.25 ± 4.11 vs. 1.36 ± 5.43 for ADIPO-IR, p = 0.001; -0.53 ± 1.88 vs. 0.03 ± 2.36 for VAI, p = 0.074. Changes in NAFLD indices were similar with different doses of pioglitazone (15, 30, or 45 mg/day), and were independent of blood glucose control. Conclusions: One-year treatment with pioglitazone even at low dosage significantly improved liver steatosis and inflammation, systemic and adipose tissue insulin resistance in patients with T2D. The beneficial effects of pioglitazone on NAFLD were independent of blood glucose control.

Objective Phthalates are non-persistent pollutants related to impaired metabolism and high cardiovascular risk. Their toxic metabolites are eliminated through urine and feces. Prevention policies are considered by the governments, although no therapeutic strategy to facilitate their elimination from the human body has been proposed so far. Aim of the present study was to verify, for the first time in humans, whether diuretics might be able to enhance phthalates’ toxic metabolites urinary output. Design and methods We conducted a two-armed, parallel-design, randomized clinical trial. Thirty patients with type 2 diabetes and hypertension received a four week-treatment with Dapagliflozin 10 mg or Hydrochlorothiazide 12.5 mg. 24-hours urine were collected to measure urinary excretion of three major 2-ethylhexyl-phthalate (DEHP) metabolites, i.e. mono 2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP) and mono 2-ethyl-5-hydroxyhexyl phthalate (MEHHP). Results 24-h urinary excretion of DEHP and MEHP was increased (+44%, p = 0.036; +49%, p = 0.0016) while MEOHP e MEHHP showed only a positive trend (+25%, p = 0.016; +36%, p = 0.062). Irrespective of the specific treatment, induced variations of daily urinary eliminations of MEHP metabolites were related with the 24-h urinary sodium (r = 0.42, p = 0.0226) and potassium (r = 0.54, p = 0.0026) excretion. Also, DEHP and MEOHP were related to sodium (r = 0·43, p = 0.0205; r = 0·44, p = 0.0168 respectively) but not to potassium. Conclusions Urinary phthalates excretion seems to occur mainly through sodium- and potassium-related mechanisms, apparently independent from the different diuretic effect. Both thiazide diuretics and SLGT2 inhibitors are effective into the removal of phthalates metabolites from the human body, reducing the human tissues’ exposure to their toxicity.

Background: Pathogenetic mechanisms involved in the progression of non-alcoholic fatty liver disease (NAFLD) are complex and multifactorial. We investigated oxidative stress through the measurement of selenoprotein P (SeP) in serum and we explored its relation to metabolic derangements and liver damage in a group of non-diabetic NAFLD subjects. Methods: 57 NAFLD patients underwent a double-tracer oral glucose tolerance test (OGTT). Insulin resistance (IR) components were calculated at baseline as follows: hepatic-IR = (endogenous glucose production*insulin), peripheral-IR = (glucose rate of disappearance(Rd)), adipose-tissue(AT)-IR as Lipo-IR = (glycerol rate of appearance (Ra)*insulin) or AT-IR = (free fatty acids (FFAs)*insulin). The lipid and amino acid (AA) profiles were assessed by gas chromatography&ndash, mass spectrometry. SeP levels were measured by enzyme immunosorbent assay. Results: Circulating SeP correlated with insulin (rS = 0.28), FFAs (rS = 0.42), glucose Rd (rS = &minus, 0.33) and glycerol Ra (rS = &minus, 0.34), consistently, SeP levels correlated with Lipo-IR and AT-IR (rS &gt, 0.4). Among the AA and lipid profiles, SeP inversely correlated with serine (rS = &minus, 0.31), glycine (rS = &minus, 0.44) and branched chain AA (rS = &minus, 0.32), and directly correlated with saturated (rS = 0.41) and monounsaturated FFAs (rS = 0.40). Hepatic steatosis and fibrosis increased in subjects with higher levels of SeP. In multivariable regression analysis, SeP was associated with the degree of hepatic fibrosis (t = 2.4, p = 0.022). Conclusions: SeP levels were associated with an altered metabolic profile and to the degree of hepatic fibrosis, suggesting a role in the pathogenesis of NAFLD.

Context Recent epidemiological observations have reported an association among phthalates exposure and insulin resistance, obesity, and diabetes but have not related exposure to these environmental pollutants with microvascular complications of diabetes. Objective To explore the relationship between phthalates and renal function in subjects with diabetes. Design Cross-sectional, case-only study. Concentrations of three urinary metabolites of di-2-ethylhexylphthalate were quantified in a spot morning urine sample, normalized for creatinine urinary excretion, and related to clinical parameters and phenotype, adjusting for confounders. Patients Two hundred and nine patients with diabetes consecutively referred to our clinic. Main outcome measures Relationship between phthalates and renal function [evaluated with estimated glomerular filtration rate (eGFR) and albuminuria]. Results Creatinine-adjusted urinary concentrations of mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP), and mono-2-ethyl-5-hydroxyhexyl phthalate were 7.53 µg/g (range, 4.84 to 12.60), 3.04 µg/g (range, 1.03 to 5.14), and 10.70 µg/g (7.02 to 17.40), respectively. Age, sex, body mass index, diabetes duration, smoking, blood pressure, glycated Hb, and eGFR did not influence their levels. Exposure to MEHP and MEOHP was greater in individuals with microalbuminuria/macroalbuminuria (MEHP, P = 0.0173; MEOHP, P = 0.0306). The fourth vs first quartile showed a greater risk of albuminuria (MEHP: OR, 4.83; 95% CI, 1.45 to 16.06; P = 0.0297; MEOHP: OR, 3.29; 95% CI, 1.08 to 10.04); P = 0.0352). MEOHP was greater (P = 0.034) in subjects with cardiovascular events; MEHP showed a positive trend (P = 0.061). Conclusion Our findings have revealed an association between exposure to di-2-ethylhexylphthalate metabolites and the degree of albuminuria in subjects with diabetes; the lack of a relationship with eGFR suggests their urinary levels are independent of renal function.

Plasma concentrations of amino acids (AAs), in particular, branched chain AAs (BCAAs), are often found increased in nonalcoholic fatty liver disease (NAFLD); however, if this is due to increased muscular protein catabolism, obesity, and/or increased insulin resistance (IR) or impaired tissue metabolism is unknown. Thus, we evaluated a) if subjects with NAFLD without obesity (NAFLD-NO) compared to those with obesity (NAFLD-Ob) display altered plasma AAs compared to controls (CTs); and b) if AA concentrations are associated with IR and liver histology. Glutamic acid, serine, and glycine concentrations are known to be altered in NAFLD. Because these AAs are involved in glutathione synthesis, we hypothesized they might be related to the severity of NAFLD. We therefore measured the AA profile of 44 subjects with NAFLD without diabetes and who had a liver biopsy (29 NAFLD-NO and 15 NAFLD-Ob) and 20 CTs without obesity, by gas chromatography-mass spectrometry, homeostasis model assessment of insulin resistance, hepatic IR (Hep-IR; Hep-IR = endogenous glucose production × insulin), and the new glutamate-serine-glycine (GSG) index (glutamate/[serine + glycine]) and tested for an association with liver histology. Most AAs were increased only in NAFLD-Ob subjects. Only alanine, glutamate, isoleucine, and valine, but not leucine, were increased in NAFLD-NO subjects compared to CTs. Glutamate, tyrosine, and the GSG-index were correlated with Hep-IR. The GSG-index correlated with liver enzymes, in particular, gamma-glutamyltransferase (R = 0.70), independent of body mass index. Ballooning and/or inflammation at liver biopsy were associated with increased plasma BCAAs and aromatic AAs and were mildly associated with the GSG-index, while only the new GSG-index was able to discriminate fibrosis F3-4 from F0-2 in this cohort. Conclusion Increased plasma AA concentrations were observed mainly in subjects with obesity and NAFLD, likely as a consequence of increased IR and protein catabolism. The GSG-index is a possible marker of severity of liver disease independent of body mass index. (Hepatology 2018;67:145-158).

The Bis(2-ethylhexyl)phthalate (DEHP), a widespread plasticizer, is considered an endocrine disrupting chemical with main toxicological effects on reproductive and metabolic systems. Human biomonitoring (HBM) studies are promoted to evaluate the background exposure levels. In the frame of LIFE PERSUADED project, the HBM study measured DEHP main metabolites (mono-(2-ethylhexyl) phthalate, MEHP; 2-ethyl-5-hydroxy-hexylphthalate, MEHHP; 2-ethyl-5-oxo-hexylphthalate, MEOHP) in Italian children and adolescent (4–14 years old) according to geographical macro-areas and areas, age and sex. Children from the South and the Centre of Italy showed higher median levels of DEHP, as a sum of its metabolites (48.14 and 47.80 μg/L), than those from the North (39.47 μg/L; p = 0.0090 and 0.0004, respectively). Considering the total population, boys are more exposed than girls (only as urinary volume), and children aged 4–6 years have higher median levels than those 7–10 and 11–14 years old. The derived reference values (RV95) for DEHP in children is 168 μg/L. The relative metabolic rates of DEHP, the background levels and, thus, the RV95, vary with the geographical area, age and sex, indicating that all these parameters should be considered in the risk assessment.

OBJECTIVE Patients with chronic liver disease (CLD), both non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis C (CHC), are at high risk of diabetes (T2D), but mechanisms are still unknown. Muscle/liver insulin resistance (IR) and pancreatic dysfunction are the major metabolic defects leading to T2D. However, if the risk of T2D in CLD patients is because of reduced insulin response and/or to IR, and the impact of liver histology has not been investigated. DESIGN We studied 220 non-T2D patients with chronic liver disease (129 NAFLD, BMI = 27.3 kg/m2 ; 91 CHC, BMI = 25.0 kg/m2 ) that received a 75-gram oral glucose tolerance test (OGTT) with the measurement of glucose and insulin concentrations for 2 hours, glucose tolerance (NGT vs IGT) and liver biopsy. The results were compared to 26 controls (CT-NGT, BMI = 25.6 kg/m2 ). We evaluated peripheral insulin sensitivity (OGIS), OGTT-insulin response (ΔAUC-I/ΔAUC-G) and disposition-index (DI = OGIS∙ΔAUC-I/ΔAUC-G) for the risk to develop T2D. RESULTS NAFLD had increased muscle IR (associated to NASH, steatosis and fibrosis), higher than in CHC or CT-NGT (OGIS = 8.9 vs 11.3 and 10.5 mL/min kg, P < .0001). In NAFLD, OGTT-insulin response (ΔAUC-I/ΔAUC-G) was the highest while it was significantly decreased in CHC (2.2 vs 1.1 and 1.6, NAFLD vs. CHC and CT-NGT, P < .005). The highest T2D risk (low DI) was observed in CHC-IGT (7.5), CHC-NGT (13.5) and NAFLD-IGT (10.8) vs CT-NGT (14.9, all P < .0001), but not in NAFL-NGT or NASH-NGT. CONCLUSION We observed an increased T2D risk in NAFLD-IGT, CHC-IGT and CHC-NGT mainly because of reduced OGTT-insulin response, while insulin response in NAFLD-NGT compensates the IR thus maintaining normal glycaemia.

Background: Naturally occurring variation in Membrane-bound O-acyltransferase domain-containing 7 (MBOAT7), encoding for an enzyme involved in phosphatidylinositol acyl-chain remodelling, has been associated with fatty liver and hepatic disorders. Here, we examined the relationship between hepatic Mboat7 down-regulation and fat accumulation. Methods: Hepatic MBOAT7 expression was surveyed in 119 obese individuals and in experimental models. MBOAT7 was acutely silenced by antisense oligonucleotides in C57Bl/6 mice, and by CRISPR/Cas9 in HepG2 hepatocytes. Findings: In obese individuals, hepatic MBOAT7 mRNA decreased from normal liver to steatohepatitis, independently of diabetes, inflammation and MBOAT7 genotype. Hepatic MBOAT7 levels were reduced in murine models of fatty liver, and by hyper-insulinemia. In wild-type mice, Mboat7 was down-regulated by refeeding and insulin, concomitantly with insulin signalling activation. Acute hepatic Mboat7 silencing promoted hepatic steatosis in vivo and enhanced expression of fatty acid transporter Fatp1. MBOAT7 deletion in hepatocytes reduced the incorporation of arachidonic acid into phosphatidylinositol, consistently with decreased enzymatic activity, determining the accumulation of saturated triglycerides, enhanced lipogenesis and FATP1 expression, while FATP1 deletion rescued the phenotype. Interpretation: MBOAT7 down-regulation by hyper-insulinemia contributes to hepatic fat accumulation, impairing phosphatidylinositol remodelling and up-regulating FATP1. Funding: LV was supported by MyFirst Grant AIRC n.16888, Ricerca Finalizzata Ministero della Salute RF-2016–02,364,358, Ricerca corrente Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; LV and AG received funding from the European Union Programme Horizon 2020 (No. 777,377) for the project LITMUS-“Liver Investigation: Testing Marker Utility in Steatohepatitis”. MM was supported by Fondazione Italiana per lo Studio del Fegato (AISF) ‘Mario Coppo’ fellowship. Keywords: LPIAT1, NAFLD, Nash, Nonalcoholic fatty liver disease, Steatohepatitis, Phospholipid, Phosphatidylinositol

Metabolomics/lipidomics are important tools to identify novel biomarkers associated with liver damage. Patients with chronic liver disease (CLD) and hepatitis C virus (HCV) infection often have alterations in glucose, lipid and protein metabolism. The aim of this study was to evaluate if dysfunctional lipid and amino acid metabolism was associated with fibrosis severity and insulin resistance in CLD/HCV patients. We analyzed the baseline sera of 75 subjects with CLD/HCV infection HCV genotype-1, with proven liver biopsy prior to antiviral treatment. We measured amino acid (AA) and lipid concentration by gas and liquid chromatography-mass spectrometry respectively. Alterations in peripheral glucose metabolism due to insulin resistance (IR) were assesed by HOMA-IR (Glucose x Insulin/22.5), while adipose tissue IR was estimated as (Adipo-IR = Free Fatty Acids x Insulin). Baseline HOMA-IR and Adipo-IR were related to the degree of liver fibrosis. Reduction in ceramides 18:1/22:0, 18:1/24:0, diacylglycerol 42:6 and increased phosphocholine 40:6 were associated with higher fibrosis. Adipo-IR was related to lower levels of lysophosphatidylcholine 14:0 and 18:2 and with higher levels of sphingomyelin 18:2/24:0 and 18:2/24:1. Almost all AA were positively associated with Adipo-IR but not with HOMA-IR. We further confirmed the potential use of metabolomics and lipidomics in CLD/HCV subjects finding novel biomarkers of hepatic fibrosis and show that the adipose tissue IR is associated with more severe liver disease and is an important marker not only of altered lipid but also AA metabolism.

Background & Aims: Naturally occurring variation in Membrane-bound O-acyltransferase domain-containing 7 (MBOAT7), an enzyme involved in phospholipid acyl-chain remodeling, has been associated with steatosis and hepatic disorders. We examined the relationship between hepatic MBOAT7 down-regulation and fat accumulation. Methods: Hepatic MBOAT7 expression was surveyed in 119 obese individuals and in experimental models. MBOAT7 was acutely silenced by antisense oligonucleotides in C57Bl/6 mice, and by CRISPR/Cas9 in HepG2. Results: In obese individuals, hepatic MBOAT7 mRNA decreased from normal liver to steatohepatitis, independently of diabetes, inflammation and MBOAT7 genotype. Mboat7 levels were reduced in murine models of fatty liver, and by hyperinsulinemia. In wild-type mice, Mboat7 was down-regulated by refeeding and insulin, concomitantly with insulin signaling activation. Acute hepatic Mboat7 silencing promoted hepatic steatosis in vivo and enhanced expression of fatty acid transporter Fatp1. MBOAT7-/- HepG2 showed altered phospholipids remodeling consistent with decreased enzymatic activity, increased triglyceride accumulation, enhanced lipogenesis and FATP1 expression, while FATP1 deletion rescued the phenotype. Conclusions: These results suggest that MBOAT7 down-regulation by hyperinsulinemia contributes to hepatic fat accumulation, impairing phospholipids remodeling and up-regulating FATP1. Funding Statement: LV was supported by MyFirst Grant AIRC n.16888, Ricerca Finalizzata Ministero della Salute RF-2016-02364358, Ricerca corrente Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, LV and AG. received funding from the European Union (EU) Programme Horizon 2020 (under grant agreement No. 777377) for the project LITMUS-“ Liver Investigation: Testing Marker Utility in Steatohepatitis”. MM was supported by Fondazione Italiana per lo Studio del Fegato (AISF) ‘Prof. Mario Coppo’ fellowship. AG received funding from the European Union (EU) Programme Horizon 2020 under grant agreement no. 634413 for the project EPoS-“Elucidating Pathways of Steatohepatitis”. Declaration of Interests: The authors declare no conflict of interest relevant to the present manuscript. Ethics Approval Statement: Informed written consent was obtained from each patient and the study protocol was approved by the Ethical Committee of the Fondazione IRCCS Ca’ Granda of Milan and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. We received an approval from an Animal Use Committee for the animal work from the University of Milan and the Italian Ministry of Health Review Boards, in the context of the protocol 8/14, as indicated in the material and methods section in the manuscript. The authorization number of this protocol is 295/2012–A, received on 12/20/2012.

Background & Aims: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is likely due to the interaction between a deranged metabolic milieu and local mediators of hepatic inflammation and fibrosis. We undertook this study to elucidate the interplay between macrophage activation, insulin resistance (IR) in target organs/tissues and hepatic damage. Methods: In 40 non-diabetic patients with biopsy-proven NAFLD we assessed: i) endogenous glucose production (EGP), glucose clearance and indexes of IR in the adipose tissue (Adipo-IR and Lipo-IR) and in the liver (Hep-IR) by tracer infusion ([6,6-2H2]glucose and [2H5]glycerol); ii) macrophage activity (by soluble sCD163) and iii) hepatic expression of CD163 (hCD163). Results: We found that sCD163 levels paralleled both the plasma free fatty acid (FFA) levels and lipolysis from adipose tissue. Consistently, sCD163 significantly correlated with adipose tissue IR (Adipo-IR: r = 0.32, p = 0.042; Lipo-IR: r = 0.39, p = 0.012). At multiple regression analysis, sCD163 levels were associated with FFA levels (r p = 0.35, p = 0.026). In vitro exposure of human monocyte-derived macrophages to palmitate enhanced sCD163 secretion. Conversely, sCD163 did not correlate with EGP or with Hep-IR. In the liver, hCD163 positively correlated with sCD163 (r = 0.58, p = 0.007) and the degree of steatosis (r = 0.34, p = 0.048), but not with EGP or Hep-IR (r = −0.27 and r = 0.11, respectively, p >0.10, both). Conclusions: Our findings suggest a link between deranged metabolism in the adipose tissue and activation of hepatic macrophages in patients with NAFLD, possibly in response to FFA overflow and independent of obesity and diabetes. Conversely, our findings do not support a link between activated hepatic macrophages and glucose metabolism (EGP or Hep-IR). The relationship between adipose tissue IR and hepatic macrophages should be considered to define therapeutic targets for NAFLD. Lay summary: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is likely due to the interaction between a deranged metabolic milieu and local mediators of hepatic inflammation and fibrosis in the insulin resistant state. This study provides in vivo support for a possible link between deranged metabolism in the adipose tissue and activation of hepatic macrophages in patients with NAFLD, most likely in response to free fatty acid overflow and independent of obesity and diabetes.

Excessive accumulation of lipids can lead to lipotoxicity, cell dysfunction and alteration in metabolic pathways, both in adipose tissue and peripheral organs, like liver, heart, pancreas and muscle. This is now a recognized risk factor for the development of metabolic disorders, such as obesity, diabetes, fatty liver disease (NAFLD), cardiovascular diseases (CVD) and hepatocellular carcinoma (HCC). The causes for lipotoxicity are not only a high fat diet but also excessive lipolysis, adipogenesis and adipose tissue insulin resistance. The aims of this review are to investigate the subtle balances that underlie lipolytic, lipogenic and oxidative pathways, to evaluate critical points and the complexities of these processes and to better understand which are the metabolic derangements resulting from their imbalance, such as type 2 diabetes and non alcoholic fatty liver disease.

Excess caloric intake does not always translate to an expansion of the subcutaneous adipose tissue (SAT) and increase in fat mass. It is now recognized that adipocyte type (white, WAT, or brown, BAT), size (large vs. small) and metabolism are important factors for the development of cardiometabolic diseases. When the subcutaneous adipose tissue is not able to expand in response to increased energy intake the excess substrate is stored as visceral adipose tissue or as ectopic fat in tissues as muscle, liver and pancreas. Moreover, adipocytes become dysfunctional (adiposopathy, or sick fat), adipokines secretion is increased, fat accumulates in ectopic sites like muscle and liver and alters insulin signaling, increasing the demand for insulin secretion. Thus, there are some subjects that despite having normal weight have the metabolic characteristics of the obese (NWMO), while some obese expand their SAT and remain metabolically healthy (MHO). In this paper we have reviewed the recent findings that relate the metabolism of adipose tissue and its composition to metabolic diseases. In particular, we have discussed the possible role of dysfunctional adipocytes and adipose tissue resistance to the antilipolytic effect of insulin on the development of impaired glucose metabolism. Finally we have reviewed the possible role of BAT vs. WAT in the alteration of lipid and glucose metabolism and the recent studies that have tried to stimulate browning in human adipose tissue.