Clusters of adipose tissue dysfunction in adults with type 2 diabetes identify those with worse lipidomic profile despite similar glycaemic control.

AIMS: To investigate clusters of adipose tissue dysfunction, that is, with adipose tissue insulin resistance (ADIPO-IR) and large waist circumference (WC), identify a worse lipidomic profile characterised by a high proportion of lipids rich in saturated fatty acids (SFA). MATERIALS AND METHODS: Hierarchical clustering based on WC and ADIPO-IR (calculated as fasting plasma non-esterified fatty acids times fasting plasma insulin, FFA×INS), was performed in 192 adults with overweight/obesity and type 2 diabetes (T2D) treated with metformin (HbA1c = 7.8%). Free fatty acid composition and lipidomic profile were measured by mass spectrometry (GC-MS and LC-MSQTOF). Indexes of fatty acid desaturation (stearoyl-coA desaturase-1 activity, SCD116 = palmitoleic acid/palmitic acid and SCD118 = oleic acid/stearic acid) and of insulin resistance (HOMA-IR) were also calculated. RESULTS: Three clusters were identified: CL1 (ADIPO-IR = 4.9 ± 2.4 and WC = 96±7 cm, mean ± SD), CL2 (ADIPO-IR = 6.5 ± 2.5 and WC = 114 ± 7 cm), and CL3 (ADIPO-IR = 15.0 ± 4.7 and WC = 107 ± 8 cm). Insulin concentrations, ADIPO-IR, and HOMA-IR significantly increased from CL1 to CL3 (all p < 0.001), while fasting glucose concentrations, HbA1c, dietary lipids and caloric intake were similar. Moreover, CL3 showed significantly higher concentrations of monounsaturated free fatty acids, oleic and palmitoleic acids, triglycerides (TAG) rich in saturated FA and associated with de novo lipogenesis (i.e., TAG 46-50), higher SCD116, SCD118, ceramide (d18:0/18:0), and phosphatidylcholine aa(36:5) compared with CL1/CL2 (all p < 0.005). CONCLUSIONS: High ADIPO-IR and large WC identify a worse lipid profile in T2D characterised by complex lipids rich in SFA, likely due to de novo synthesis given higher plasma monounsaturated FFA and increased desaturase activity indexes. REGISTRATION NUMBER TRIAL: ID NCT00700856 https://clinicaltrials.gov.

Brain histone beta-hydroxybutyrylation couples metabolism with gene expression.

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.

Fatty Liver Index (FLI) Identifies Not Only Individuals with Liver Steatosis but Also at High Cardiometabolic Risk.

A fatty liver index (FLI) greater than sixty (FLI ≥ 60) is an established score for metabolic dysfunction-associated steatotic liver disease (MASLD), which carries a high risk for diabetes and cardiovascular disease, while a FLI ≤ 20 rules out the presence of steatosis. Thus, we investigated whether FLI was associated with cardiometabolic risk factors, i.e., visceral (VAT), subcutaneous (SC), epicardial (EPI), extrapericardial (PERI), and total cardiac (CARD-AT) adipose tissue, hepatic fat ((by magnetic resonance imaging, MRI, and spectroscopy, MRS), and insulin resistance (IR, HOMA-IR and OGIS-index), and components of metabolic syndrome. All individuals with FLI ≥ 60 had MASLD, while none with FLI ≤ 20 had steatosis (by MRS). Subjects with FLI ≥ 60 had a higher BMI and visceral and cardiac fat (VAT > 1.7 kg, CARD-AT > 0.2 kg). FLI was positively associated with increased cardiac and visceral fat and components of metabolic syndrome. FLI, VAT, and CARD-AT were all associated with IR, increased blood pressure, cholesterol, and reduced HDL. For FLI ≥ 60, the cut-off values for fat depots and laboratory measures were estimated. In conclusion, FLI ≥ 60 identified not only subjects with steatosis but also those with IR, abdominal and cardiac fat accumulation, increased blood pressure, and hyperlipidemia, i.e., those at higher risk of cardiometabolic diseases. Targeted reduction of FLI components would help reduce cardiometabolic risk.

Adipose tissue dysfunction and visceral fat are associated with hepatic insulin resistance and severity of NASH even in lean individuals.

BACKGROUND & AIMS: 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. METHODS: Thirty-two non-diabetic NAFLD with liver biopsy (BMI 21.4-34.7 kg/m2 ) and eight lean individuals (BMI 19.6-22.8 kg/m2 ) were characterized for fat distribution (VF, SC-AT and IHTG by magnetic resonance imaging), lipolysis and insulin resistance by tracer infusion, free fatty acids (FFAs) and triglyceride (TAG) concentration and composition (by mass spectrometry). RESULTS: Intrahepatic 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. CONCLUSIONS: These findings propose VF as an early indicator of NAFLD progression independently of BMI, which may allow for evidence-based prevention and intervention strategies.

Exposure to Endocrine Disruptors (Di(2-Ethylhexyl)phthalate (DEHP) and Bisphenol A (BPA)) in Women from Different Residing Areas in Italy: Data from the LIFE PERSUADED Project.

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.

PPAR-γ-induced changes in visceral fat and adiponectin levels are associated with improvement of steatohepatitis in patients with NASH.

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 < .007, respectively) and reduction in VF/SC fat (r = .41 and r = .37, P < .03 respectively). CONCLUSIONS: Reduction in VF and improved VF/SC-distribution, combined with an increase in ADPN, mediate the histological benefits of PPAR-γ action, highlighting the central role of fat metabolism and its distribution on steatohepatitis disease activity in patients with NASH.

SGLT2 inhibitors and thiazide enhance excretion of DEHP toxic metabolites in subjects with type 2 diabetes: A randomized clinical trial.

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.

Mechanisms for increased risk of diabetes in chronic liver diseases.

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.

Biomonitoring of Bis(2-ethylhexyl)phthalate (DEHP) in Italian children and adolescents: Data from LIFE PERSUADED project.

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.

Interplay between Oxidative Stress and Metabolic Derangements in Non-Alcoholic Fatty Liver Disease: The Role of Selenoprotein P.

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-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 = -0.33) and glycerol Ra (rS = -0.34); consistently, SeP levels correlated with Lipo-IR and AT-IR (rS > 0.4). Among the AA and lipid profiles, SeP inversely correlated with serine (rS = -0.31), glycine (rS = -0.44) and branched chain AA (rS = -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.

Crosstalk between adipose tissue insulin resistance and liver macrophages in non-alcoholic fatty liver disease.

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 (rp = 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.

Altered amino acid concentrations in NAFLD: Impact of obesity and insulin resistance.

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).

Altered Metabolic Profile and Adipocyte Insulin Resistance Mark Severe Liver Fibrosis in Patients with Chronic Liver Disease.

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.

Hepatic glucose metabolism in the steatotic liver.

The liver is central in regulating glucose homeostasis, being the major contributor to endogenous glucose production and the greatest reserve of glucose as glycogen. It is both a target and regulator of the action of glucoregulatory hormones. Hepatic metabolic functions are altered in and contribute to the highly prevalent steatotic liver disease (SLD), including metabolic dysfunction-associated SLD (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). In this Review, we describe the dysregulation of hepatic glucose metabolism in MASLD and MASH and associated metabolic comorbidities, and how advances in techniques and models for the assessment of hepatic glucose fluxes in vivo have led to the identification of the mechanisms related to the alterations in glucose metabolism in MASLD and comorbidities. These fluxes can ultimately increase hepatic glucose production concomitantly with fat accumulation and alterations in the secretion and action of glucoregulatory hormones. No pharmacological treatment has yet been approved for MASLD or MASH, but some antihyperglycaemic drugs approved for treating type 2 diabetes have shown positive effects on hepatic glucose metabolism and hepatosteatosis. A deep understanding of how MASLD affects glucose metabolic fluxes and glucoregulatory hormones might assist in the early identification of at-risk individuals and the use or development of targeted therapies.

GLP-1 enhances beta-cell response to protein ingestion and bariatric surgery amplifies it.

OBJECTIVE: Protein ingestion stimulates ß-cell secretion and alters glucose flux. Enhanced action of glucagon-like peptide 1 (GLP-1) and increased plasma glucose excursion contribute to prandial hyperinsulinemia after gastric bypass surgery (GB) and sleeve gastrectomy (SG). We examined the contribution of endogenous GLP-1 to glucose kinetics and ß-cell response to protein ingestion under basal glucose concentrations in humans, and whether these responses are affected by rerouted gut after GB or SG. DESIGN: Glucose fluxes, insulin secretion rate (ISR), and incretin responses to a 50-gram oral protein load were compared between 10 non-diabetic individuals with GB, 9 matched subjects with SG and 7 non-operated controls (CN) with and without intravenous infusion of exendin-(9- 39) [Ex-9), a specific GLP-1 receptor (GLP-1R) antagonist. RESULTS: Blocking GLP-1R increased the plasma glucose concentration before and after protein ingestion in all 3 groups (p<0.05) and decreased ß-cell sensitivity to glucose in the first 30 minutes of protein ingestion (p<0.05). Reduction in the prandial ISR3h by Ex-9 infusion, however, only was observed in GB and SG (p<0.05 for interaction) and not in controls. Also, GLP-1R blockade increased post-protein insulin action in GB and SG, but not CN (p=0.09 for interaction). Endogenous glucose production (EGP) during the first 60 minutes after protein ingestion was increased in all 3 groups but EGP3h only was accentuated in GB by Ex-9 infusion (p<0.05 for interaction). CONCLUSION: These findings are consistent with both a pancreatic and extrapancreatic role for GLP-1 during protein ingestion in humans, and GLP-1 actions are exaggerated by bariatric surgery.

GLP-1 Receptor Agonist Treatment Improves Fasting and Postprandial Lipidomic Profiles Independently of Diabetes and Weight Loss.

Treatment with glucagon-like peptide 1 receptor agonists reduces liver steatosis and cardiometabolic risk (CMR). Few data are available on lipid metabolism, and no information is available on the postprandial lipidomic profile. Thus, we investigated how exenatide treatment changes lipid metabolism and composition during fasting and after a mixed-meal tolerance test (MMTT) in adults with severe obesity without diabetes. Thirty individuals (26 females and 4 males, 30-60 years old, BMI >40 kg/m2, HbA1c 5.76%) were assigned (1:1) to diet with exenatide 10 µg twice daily treatment (n = 15) or without treatment as control (n = 15) for 3 months. Fasting and postprandial lipidomic profile (by liquid chromatography quadrupole time-of-flight mass spectrometry) and fatty acid metabolism (following a 6-h MMTT/tracer study) and composition (by gas chromatography-mass spectrometry) were evaluated before and after treatment. Both groups had slight weight loss (-5.5% vs. -1.9%, exenatide vs. control; P = 0.052). During fasting, exenatide, compared with control, reduced some ceramides (CERs) and lysophosphatidylcholines (LPCs) previously associated with CMR, while relatively increasing unsaturated phospholipid species (phosphatidylcholine [PC], LPC) with protective effects on CMR, although concentrations of total lipid species were unchanged. During MMTT, both groups showed suppressed lipolysis equal to baseline, but exenatide significantly lowered free fatty acid clearance and postprandial triacyclglycerol (TAG) concentrations, particularly saturated TAGs with 44-54 carbons. Exenatide also reduced some postprandial CERs, PCs, and LPCs previously linked to CMR. These changes in lipidomic profile remained statistically significant after adjusting for weight loss. Exenatide improved fasting and postprandial lipidomic profiles associated with CMR mainly by reducing saturated postprandial TAGs and CERs independently of weight loss and diabetes.

Urinary Bisphenol-A (BPA) and Bis(2-ethylhexyl)phthalate (DEHP) metabolite concentrations in children with obesity: A case-control study.

Introduction Obesity is a worldwide public health problem. Experimental animal and in vitro studies suggest that the exposure to BPA and phthalates are associated to a higher risk of obesity. Objective To determine urinary excretion of bisphenol A and phthalates in obese and normal weight children. Methods A case-control study was conducted in 122 children. Sixty-six obese children 36 girls (mean age 8.41±1.27 years) and 30 boys (mean age 8.51 ± 1.33 years), and 56 normal weight children, 27 girls (mean age 7.64 ± 1.49 years) and 29 boys (mean age 7.77 ± 1.56 years) were studied. Urinary BPA and Bis(2-ethylhexyl) phthalate (DEHP) metabolites (MEHP, MEHHP and MEOHP) were measured respectively by gas chromatography and high-performance liquid chromatography. Individual determinants of exposure were evaluated through "ad hoc" questionnaires. Results BPA and DEHP metabolites were detectable in obese and normal weight children. Obese girls showed significantly higher BPA concentrations in comparison with normal weight girls (means 10.77, 95% CI 7.02-16.53 vs 5.50, 95% CI 3.93-7.71 µg/g creatinine, respectively, p< 0.02). The first step of DEHP metabolic rate was significantly higher in obese girls compared with controls (p<0.05). DEHP metabolites correlated significantly with leptin concentrations in obese girls (p< 0.03). A higher risk of obesity was found in children with BPA levels above the median values with the habit to eat food packaged (OR=11.09, 95% CI=1.28-95.78). Conclusions These findings show that a higher exposure to BPA is associated with the risk of obesity in girls. Further studies are needed to unveil the cause-effect relationship.

Assessment of Exposure to Di-(2-ethylhexyl) Phthalate (DEHP) Metabolites and Bisphenol A (BPA) and Its Importance for the Prevention of Cardiometabolic Diseases.

Exposomics analyses have highlighted the importance of biomonitoring of human exposure to pollutants, even non-persistent, for the prevention of non-communicable diseases such as obesity, diabetes, non-alcoholic fatty liver disease, atherosclerosis, and cardiovascular diseases. Phthalates and bisphenol A (BPA) are endocrine disrupting chemicals (EDCs) widely used in industry and in a large range of daily life products that increase the risk of endocrine and cardiometabolic diseases especially if the exposure starts during childhood. Thus, biomonitoring of exposure to these compounds is important not only in adulthood but also in childhood. This was the goal of the LIFE-PERSUADED project that measured the exposure to phthalates (DEHP metabolites, MEHP, MEHHP, MEOHP) and BPA in Italian mother-children couples of different ages. In this paper we describe the method that was set up for the LIFE PERSUADED project and validated during the proficiency test (ICI/EQUAS) showing that accurate determination of urinary phthalates and BPA can be achieved starting from small sample size (0.5 mL) using two MS techniques applied in cascade on the same deconjugated matrix.

High exposure to phthalates is associated with HbA1c worsening in type 2 diabetes subjects with and without edentulism: a prospective pilot study.

BACKGROUND: Phthalates exposure and complete edentulism are related to both low socioeconomic status. No study by far has verified if and to what extent these two conditions are related. We aimed to explore their potential association and interplay in the metabolic control and cardiovascular risk profile. METHODS: In our small (n = 48) prospective pilot study twenty-four patients with type 2 diabetes (DnE) and twenty-four patients with type 2 diabetes and edentulism (DE) followed for 19 ± 2 months were treated according to best clinical standards. Phthalates' exposure was evaluated by urinary concentration of di-2-ethylhexylphthalate (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: No association between phthalates and edentulism was found, nor did edentulism affect glucose control. Higher phthalates exposure was associated with a glycated haemoglobin worsening. This association was found for all the measured phthalates metabolites, both as a whole (DEHP; r = 0.33, p = 0.0209) and individually: MEHP (r = 0.41, p = 0.0033), MEHHP (r = 0.32, p = 0.028), MEOHP (r = 0.28, p = 0.0386). CONCLUSIONS: Phthalates are not associated with edentulism but predict the worsening of glucose control in subjects with type 2 diabetes. These findings might prove relevant in identifying novel biomarkers of cardiometabolic risk. Further studies are needed to validate our results and estimate the true potential of phthalates in terms of risk assessment.

TM6SF2/PNPLA3/MBOAT7 Loss-of-Function Genetic Variants Impact on NAFLD Development and Progression Both in Patients and in In Vitro Models.

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.