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Among inflammation and coagulation markers, PAI-1 is a true component of the metabolic syndrome

Objective:To investigate whether leukocyte count, fibrinogen, von Willebrand factor (vWF) and plasminogen activator inhibitor-1 activity (PAI-1) are increased in subjects with the metabolic syndrome as defined by the National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATPIII) and the World Health Organisation (WHO).Design:Cross-sectional study.Subjects:A total of 520 overweight and obese subjects: 379 women and 141 men, visiting the weight management clinic of a University Hospital.Subjects and measurements:Waist circumference, triglycerides, HDL cholesterol, blood pressure and fasting glucose were determined, and the presence or absence of the metabolic syndrome according to the NCEP-ATPIII criteria was assessed. In 349 subjects, data on the waist-to-hip ratio (WHR) and albumin excretion rate were available and the WHO criteria were applied. Insulin resistance was defined using the HOMA-IR index.Results:Subjects with the metabolic syndrome according to the NCEP-ATPIII criteria had significantly higher levels of leukocyte count (P<0.001) and PAI-1 (P<0.001), while no significant differences were found for fibrinogen or vWF (P>0.05). Using the WHO criteria, similar results were found except for vWF, where higher levels were found in subjects with the metabolic syndrome. When subjects were classified according to the number of components of the metabolic syndrome, levels of leukocyte count, vWF and PAI-1 activity were significantly different (P<0.05). In logistic regression analysis PAI-1, gender and leukocyte count were independent determinants of the metabolic syndrome (P<0.001).Conclusion:Evidence for being a true component of the metabolic syndrome is strong for PAI-1, less for leukocyte count and weak for vWF and fibrinogen.

Role of insulin as a negative regulator of plasma endocannabinoid levels in obese and nonobese subjects

OBJECTIVE Endocannabinoids (ECs) control metabolism via cannabinoid receptors type 1 (CB1). Their plasma levels are elevated in overweight type 2 diabetes (T2D) and in obese patients, and decrease postprandially in normoweight individuals. We investigated in two different cohorts of nonobese or obese volunteers whether oral glucose in glucose tolerance tests (OGTT) or acute insulin infusion during euglycemic hyperinsulinemic clamp affect plasma EC levels. DESIGN AND METHODS OGTT was performed in ten obese hyperinsulinemic patients (body mass index (BMI)=35.8 kg/m2, fasting insulin=14.83 mU/l), and ten normoweight normoinsulinemic volunteers (BMI=21.9 kg/m2, fasting insulin=7.2 mU/l). Insulin clamp was performed in 19 mostly nonobese men (BMI=25.8 kg/m2) with varying degrees of liver fat and plasma triglycerides (TGs), with (n=7) or without T2D. Plasma levels of ECs (anandamide and 2-arachidonoylglycerol (2-AG)) were measured by liquid chromatography-mass spectrometry, before and 60 and 180 min after OGTT, and before and 240 and 480 min after insulin or saline infusion. RESULTS Oral glucose load decreased anandamide plasma levels to an extent inversely correlated with BMI, waist circumference, subcutaneous fat, fasting insulin and total glucose, and insulin areas under the curve during the OGTT, and nonsignificantly in obese volunteers. Insulin infusion decreased anandamide levels to an extent that weakly, but significantly, correlated negatively with TGs, liver fat and fasting insulin, and positively with high density lipoprotein cholesterol. OGTT decreased 2-AG levels to a lower extent and in a way weakly inversely correlated with fasting insulin. CONCLUSIONS We suggest that insulin reduces EC levels in a way inversely related to anthropometric and metabolic predictors of insulin resistance and dyslipidemia.

Transcriptional Activation of Apolipoprotein CIII Expression by Glucose May Contribute to Diabetic Dyslipidemia

Objective—Hypertriglyceridemia and fatty liver are common in patients with type 2 diabetes, but the factors connecting alterations in glucose metabolism with plasma and liver lipid metabolism remain unclear. Apolipoprotein CIII (apoCIII), a regulator of hepatic and plasma triglyceride metabolism, is elevated in type 2 diabetes. In this study, we analyzed whether apoCIII is affected by altered glucose metabolism. Methods and Results—Liver-specific insulin receptor–deficient mice display lower hepatic apoCIII mRNA levels than controls, suggesting that factors other than insulin regulate apoCIII in vivo. Glucose induces apoCIII transcription in primary rat hepatocytes and immortalized human hepatocytes via a mechanism involving the transcription factors carbohydrate response element–binding protein and hepatocyte nuclear factor-4&agr;. ApoCIII induction by glucose is blunted by treatment with agonists of farnesoid X receptor and peroxisome proliferator-activated receptor-&agr; but not liver X receptor, ie, nuclear receptors controlling triglyceride metabolism. Moreover, in obese humans, plasma apoCIII protein correlates more closely with plasma fasting glucose and glucose excursion after oral glucose load than with insulin. Conclusion—Glucose induces apoCIII transcription, which may represent a mechanism linking hyperglycemia, hypertriglyceridemia, and cardiovascular disease in type 2 diabetes.

Prothrombotic factors in histologically proven nonalcoholic fatty liver disease and nonalcoholic steatohepatitis

An independent role of nonalcoholic fatty liver disease (NAFLD) in the development of cardiovascular disease has been suggested, probably mediated through increased levels of prothrombotic factors. Therefore, we examined whether NAFLD is linked to a prothrombotic state, independently of metabolic risk factors in a large single‐center cohort of overweight/obese patients. Patients presenting to the obesity clinic underwent a detailed metabolic and liver assessment, including an extensive panel of coagulation factors. If NAFLD was suspected, a liver biopsy was proposed. A series of 273 consecutive patients (65% female) with a liver biopsy were included (age, 44 ± 0.76 years; body mass index: 39.6 ± 0.40 kg/m2). Increase in fibrinogen, factor VIII, and von Willebrand factor and decrease in antithrombin III correlated with metabolic features, but not with liver histology. Levels of plasminogen activator inhibitor‐1 (PAI‐1) increased significantly with increasing severity of steatosis (P < 0.001), lobular inflammation (P < 0.001), ballooning (P = 0.002), and fibrosis (P < 0.001). Patients with nonalcoholic steatohepatitis had significantly higher PAI‐1 values than those with normal liver (P < 0.001). In multiple regression, including anthropometric and metabolic parameters, steatosis remained an independent predictor of PAI‐1 levels, explaining, together with fasting C‐peptide and waist circumference, 21% of the variance in PAI‐1. No consistent correlations with histology were found for the other coagulation factors. Conclusion: In obesity, NAFLD severity independently contributes to the increase in PAI‐1 levels, whereas other coagulation factors are unaltered. This finding might, in part, explain the increased cardiovascular risk associated with NAFLD. (Hepatology 2014;58:121–129)

PPARα gene expression correlates with severity and histological treatment response in patients with non-alcoholic steatohepatitis

BACKGROUND & AIMS Peroxisome proliferator-activated receptors (PPARs) have been implicated in non-alcoholic steatohepatitis (NASH) pathogenesis, mainly based on animal data. Gene expression data in NASH patients are scarce. We studied liver PPARα, β/δ, and γ expression in a large cohort of obese patients assessed for presence of NAFLD at baseline and 1 year follow-up. METHODS Patients presented to the obesity clinic underwent a hepatic work-up. If NAFLD was suspected, liver biopsy was performed. Gene expression was studied by mRNA quantification. Patients were reassessed after 1 year. RESULTS 125 patients were consecutively included in the study, of which 85 patients had paired liver biopsy taken at 1 year of follow-up. Liver PPARα expression negatively correlated with the presence of NASH (p=0.001) and with severity of steatosis (p=0.003), ballooning (p=0.001), NASH activity score (p=0.008) and fibrosis (p=0.003). PPARα expression was positively correlated to adiponectin (R(2)=0.345, p=0.010) and inversely correlated to visceral fat (R(2)=-0.343, p<0.001), HOMA IR (R(2)=-0.411, p<0.001) and CK18 (R(2)=-0.233, p=0.012). Liver PPARβ/δ and PPARγ expression did not correlate with any histological feature nor with glucose metabolism or serum lipids. At 1 year, correlation of PPARα expression with liver histology was confirmed. In longitudinal analysis, an increase in expression of PPARα and its target genes was significantly associated with histological improvement (p=0.008). CONCLUSION Human liver PPARα gene expression negatively correlates with NASH severity, visceral adiposity and insulin resistance and positively with adiponectin. Histological improvement is associated with an increase in expression of PPARα and its target genes. These data might suggest that PPARα is a potential therapeutic target in NASH.

Prediction of non-alcoholic fatty-liver disease and liver fat content by serum molecular lipids

Aims/hypothesisWe examined whether analysis of lipids by ultra-performance liquid chromatography (UPLC) coupled to MS allows the development of a laboratory test for non-alcoholic fatty-liver disease (NAFLD), and how a lipid-profile biomarker compares with the prediction of NAFLD and liver-fat content based on routinely available clinical and laboratory data.MethodsWe analysed the concentrations of molecular lipids by UPLC-MS in blood samples of 679 well-characterised individuals in whom liver-fat content was measured using proton magnetic resonance spectroscopy (1H-MRS) or liver biopsy. The participants were divided into biomarker-discovery (n = 287) and validation (n = 392) groups to build and validate the diagnostic models, respectively.ResultsIndividuals with NAFLD had increased triacylglycerols with low carbon number and double-bond content while lysophosphatidylcholines and ether phospholipids were diminished in those with NAFLD. A serum-lipid signature comprising three molecular lipids (‘lipid triplet’) was developed to estimate the percentage of liver fat. It had a sensitivity of 69.1% and specificity of 73.8% when applied for diagnosis of NAFLD in the validation series. The usefulness of the lipid triplet was demonstrated in a weight-loss intervention study.Conclusions/interpretationThe liver-fat-biomarker signature based on molecular lipids may provide a non-invasive tool to diagnose NAFLD, in addition to highlighting lipid molecular pathways involved in the disease.

C-reactive protein levels in relation to various features of non-alcoholic fatty liver disease among obese patients

BACKGROUND & AIMS Non-alcoholic fatty liver disease (NAFLD) is a major hepatic consequence of obesity. It has been suggested that the high sensitivity C-reactive protein (hs-CRP) is an obesity-independent surrogate marker of severity of NAFLD, especially development of non-alcoholic steato-hepatitis (NASH), but this remains controversial. We aimed to investigate whether associations between various features of NAFLD and hs-CRP are independent of body mass index (BMI) in its broad range among obese patients. METHODS A total of 627 obese adults (80% females), representing three cohorts from France and Belgium, had information on liver histology obtained from liver biopsies and measures of hs-CRP and BMI. We investigated whether the different features of NAFLD and BMI were associated with hs-CRP, with and without mutual adjustments using linear regression. RESULTS BMI and hs-CRP were strongly associated. Per every 10% increase in BMI the hs-CRP level increased by 19-20% (p<0.001), and adjustment for NAFLD-stage (including no-NAFLD) did not influence the association. We found no BMI-independent association between NASH and hs-CRP. However, a positive association between degree of steatosis and hs-CRP was observed (p<0.05) and this effect remained significant after adjusting for BMI, lobular inflammation, hepatocyte ballooning, and fibrosis. We found no significant associations between the other features of NAFLD and hs-CRP. CONCLUSIONS This study indicates that it is the accumulation of fat -both in the adipose tissue and in liver steatosis- that leads to increased hs-CRP levels among obese patients. Thus, hs-CRP may be a marker of steatosis, but not of severity of NAFLD, in obese patients.

Visceral adipose tissue and inflammation correlate with elevated liver tests in a cohort of overweight and obese patients

Objective:To study the relationship between elevated liver tests and high sensitive C-reactive protein (hs-CRP), as potential markers of liver inflammation and non-alcoholic steatohepatitis (NASH), with anthropometric and laboratory parameters in overweight patients, especially the relationship with visceral adipose tissue (VAT).Methods:Patients presenting to the obesity clinic were prospectively included. Detailed anthropometry, computed tomography (CT)-measured VAT, liver tests (aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT)) and hs-CRP were assessed, along with an extended series of biochemical parameters.Results:All 480 patients (gender distribution male (M)/female (F) (10/90%)) with complete data were included. Mean age was 39±13 years, mean BMI 34.5±6.0 kg m−2. In 37.3% of the patients one or more of the liver tests were elevated. VAT was positively related to AST (r=0.18, P<0.001), ALT (r=0.29, P<0.001), ALP (r=0.16, P<0.01) and GGT (r=0.39, P<0.001). Comparing subjects with high (VAT⩾113 cm2) vs low (VAT<113 cm2) VAT levels, significant differences were noted for AST (26±12 vs 24±12 U l−1, P=0.003), ALT (37±21 vs 31±21 U l−1, P<0.001), ALP (76±20 vs 71±18 U l−1, P=0.008), GGT (33±20 vs 25±15 U l−1, P<0.001) and hs-CRP (0.62±0.43 vs 0.52±0.48 mg dl−1, P<0.001). After correction for BMI the difference in AST and ALP between the high vs low VAT group disappeared. The differences for ALT and GGT remained significant (P=0.008 and P<0.001 respectively). After correction for hs-CRP the four different liver tests remained significantly higher in the high VAT group. A stepwise multiple regression analysis revealed that every single liver test has his own most important determinant; VAT and hs-CRP for AST, insulin resistance calculated with homeostasis model assessment (HOMA-IR) and hs-CRP for ALT and ALP, and triglycerides and VAT for GGT.Conclusion:In overweight and obese patients, liver tests, especially ALT and GGT, are associated with visceral fat mass. After correction for BMI and hs-CRP, ALT and GGT are significantly higher in patients with increased VAT, thereby supporting evidence for a potential key role of VAT in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).

Noninvasive assessment of nonalcoholic fatty liver disease in obese or overweight patients.

BACKGROUND & AIMS Reliable noninvasive tools are needed for staging nonalcoholic fatty liver disease (NAFLD). Published scoring systems have not been validated in prospective assessments of unselected patients. We aimed to identify factors that predicted development of nonalcoholic steatohepatitis (NASH) in a large group of overweight or obese patients and compared these with established factors. METHODS We performed a prospective analysis of factors associated with the development and severity of NAFLD in patients at a single obesity center. We evaluated liver involvement in 542 patients by a large set of routine and non-routine parameters, including ultrasound and genetic testing. Those suspected of having NAFLD underwent liver biopsy (57.7%). Patients were divided into design cohort (n = 200) and validation cohort (n = 113) to identify factors associated with the presence and severity of NAFLD and NASH. RESULTS Factors independently associated with development of NASH included increased levels of alanine aminotransferase (ALT), fasting levels of C-peptide, and ultrasound steatosis scores (USSs), with area under the receiver operating curve (AUROC) values of 0.854 in the design cohort and 0.823 in the validation cohort. NASH activity scores also correlated with level of ALT, USS, and fasting level of C-peptide (R(2) = 0.491). Independent predictors of advanced fibrosis included waist circumference and level of aspartate aminotransferase (AUROC values of 0.839 and 0.846 for design and validation cohorts, respectively; negative predictive values of 98% and 97%, respectively, for a cutoff of -2.14). Previously published scoring systems had significantly lower AUROC values. Levels of CK18 and PNPLA3 polymorphisms correlated with development of NASH but did not add value. CONCLUSIONS Parameters routinely analyzed in assessing obese patients can be used to determine the presence of NASH and advanced fibrosis. Non-routine tests do not increase diagnostic accuracy. Previously published scores are significantly less accurate.

Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease

The liver is a major target of injury in obese patients. Non-alcoholic fatty liver disease (NAFLD) is present in 60-90% of obese Americans and can range from simple steatosis to the more severe non-alcoholic steatohepatitis (NASH). The onset of a chronic inflammatory reaction marks the progression from simple steatosis to NASH and the expansion of adipose tissue is strongly associated with angiogenesis. Therefore, we determined the serum concentration of inflammatory [tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6)] and angiogenic [vascular endothelial growth factor A (VEGF)] cytokines and soluble VEGF receptors 1 and 2 (sVEGFR1, sVEGFR2) in the serum of an obese population with simple steatosis and NASH compared to healthy controls. Moreover, we determined the TNFα, IL6, VEGF, VEGFR1 and VEGFR2 gene expression in the liver of these simple steatosis and NASH patients. The population consisted of 30 obese patients, which were diagnosed with simple steatosis and 32 patients with NASH and compared to 30 age-and-sex matched healthy controls. Mean serum TNFα levels were elevated in the serum of simple steatosis and NASH patients compared to healthy controls, reaching significance in NASH patients. IL6 was significantly increased in simple steatosis and NASH patients compared to the healthy controls. VEGF levels were significantly elevated in patients with simple steatosis and borderline significantly elevated in NASH patients compared to the serum levels of healthy control subjects. The concentration of sVEGFR1 was significantly increased in serum of simple steatosis and NASH patients compared to controls. sVEGFR2 concentration was not significantly different in the three groups. TNFα mRNA expression was higher in NASH patients compared to simple steatosis patients. Hepatic gene expression of VEGF, VEGFR1 and VEGFR2 were slightly decreased in NASH patients compared to simple steatosis patients. These data indicate the involvement of inflammatory (TNFα and IL6), angiogenic (VEGF) cytokines and sVEGFR1 in the pathophysiology of NAFLD.