Ksenia Sevastianova

Prediction of Non-Alcoholic Fatty Liver Disease and Liver Fat Using Metabolic and Genetic Factors

BACKGROUND & AIMS Our aims were to develop a method to accurately predict non-alcoholic fatty liver disease (NAFLD) and liver fat content based on routinely available clinical and laboratory data and to test whether knowledge of the recently discovered genetic variant in the PNPLA3 gene (rs738409) increases accuracy of the prediction. METHODS Liver fat content was measured using proton magnetic resonance spectroscopy in 470 subjects, who were randomly divided into estimation (two thirds of the subjects, n = 313) and validation (one third of the subjects, n = 157) groups. Multivariate logistic and linear regression analyses were used to create an NAFLD liver fat score to diagnose NAFLD and liver fat equation to estimate liver fat percentage in each individual. RESULTS The presence of the metabolic syndrome and type 2 diabetes, fasting serum (fS) insulin, fS-aspartate aminotransferase (AST), and the AST/alanine aminotransferase ratio were independent predictors of NAFLD. The score had an area under the receiver operating characteristic curve of 0.87 in the estimation and 0.86 in the validation group. The optimal cut-off point of -0.640 predicted increased liver fat content with sensitivity of 86% and specificity of 71%. Addition of the genetic information to the score improved the accuracy of the prediction by only <1%. Using the same variables, we developed a liver fat equation from which liver fat percentage of each individual could be estimated. CONCLUSIONS The NAFLD liver fat score and liver fat equation provide simple and noninvasive tools to predict NAFLD and liver fat content.

FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies: a diagnostic study

BACKGROUND Muscle biopsy is the gold standard for diagnosis of mitochondrial disorders because of the lack of sensitive biomarkers in serum. Fibroblast growth factor 21 (FGF-21) is a growth factor with regulatory roles in lipid metabolism and the starvation response, and concentrations are raised in skeletal muscle and serum in mice with mitochondrial respiratory chain deficiencies. We investigated in a retrospective diagnostic study whether FGF-21 could be a biomarker for human mitochondrial disorders. METHODS We assessed samples from adults and children with mitochondrial disorders or non-mitochondrial neurological disorders (disease controls) from seven study centres in Europe and the USA, and recruited healthy volunteers (healthy controls), matched for age where possible, from the same centres. We used ELISA to measure FGF-21 concentrations in serum or plasma samples (abnormal values were defined as >200 pg/mL). We compared these concentrations with values for lactate, pyruvate, lactate-to-pyruvate ratio, and creatine kinase in serum or plasma and calculated sensitivity, specificity, and positive and negative predictive values for all biomarkers. FINDINGS We analysed serum or plasma from 67 patients (41 adults and 26 children) with mitochondrial disorders, 34 disease controls (22 adults and 12 children), and 74 healthy controls. Mean FGF-21 concentrations in serum were 820 (SD 1151) pg/mL in adult and 1983 (1550) pg/mL in child patients with respiratory chain deficiencies and 76 (58) pg/mL in healthy controls. FGF-21 concentrations were high in patients with mitochondrial disorders affecting skeletal muscle but not in disease controls, including those with dystrophies. In patients with abnormal FGF-21 concentrations in serum, the odds ratio of having a muscle-manifesting mitochondrial disease was 132·0 (95% CI 38·7-450·3). For the identification of muscle-manifesting mitochondrial disease, the sensitivity was 92·3% (95% CI 81·5-97·9%) and specificity was 91·7% (84·8-96·1%). The positive and negative predictive values for FGF-21 were 84·2% (95% CI 72·1-92·5%) and 96·1 (90·4-98·9%). The accuracy of FGF-21 to correctly identify muscle-manifesting respiratory chain disorders was better than that for all conventional biomarkers. The area under the receiver-operating-characteristic curve for FGF-21 was 0·95; by comparison, the values for other biomarkers were 0·83 lactate (p=0·037, 0·83 for pyruvate (p=0·015), 0·72 for the lactate-to-pyruvate ratio (p=0·0002), and 0·77 for creatine kinase (p=0·013). INTERPRETATION Measurement of FGF-21 concentrations in serum identified primary muscle-manifesting respiratory chain deficiencies in adults and children and might be feasible as a first-line diagnostic test for these disorders to reduce the need for muscle biopsy. FUNDING Sigrid Jusélius Foundation, Jane and Aatos Erkko Foundation, Molecular Medicine Institute of Finland, University of Helsinki, Helsinki University Central Hospital, Academy of Finland, Novo Nordisk, Arvo and Lea Ylppö Foundation.

Cholesterol synthesis is increased and absorption decreased in non-alcoholic fatty liver disease independent of obesity

BACKGROUND & AIMS Non-alcoholic fatty liver disease (NAFLD) is associated with impaired glucose and lipoprotein metabolism. However, the metabolism of cholesterol in NAFLD remains unexplored. We investigated how fatty liver influences cholesterol metabolism in 242 non-diabetic subjects. METHODS Liver fat content was measured with proton magnetic resonance spectroscopy. Cholesterol metabolism was assayed with serum non-cholesterol sterols, surrogate markers of cholesterol synthesis and absorption. The analyses were performed with gas-liquid chromatography. RESULTS A total of 114 subjects had NAFLD and 128 subjects had normal liver fat content. Non-cholesterol sterols reflecting cholesterol synthesis (cholestenol, desmosterol, and lathosterol) were higher, and those reflecting cholesterol absorption (cholestanol and plant sterols) were lower in subjects with NAFLD than in controls, independent of body mass index. Liver fat content was positively associated with markers of cholesterol synthesis (r = from 0.262 to 0.344, p < 0.001 for all) and inversely associated with markers of cholesterol absorption (r = from -0.299 to -0.336, p < 0.001 for all). In the entire study group, synthesis and absorption markers were interrelated, indicating that the homeostasis of cholesterol metabolism was maintained. LDL cholesterol was similar in the two groups. CONCLUSIONS We demonstrated that although LDL cholesterol concentrations are unchanged, cholesterol metabolism in NAFLD is characterized by increased synthesis and diminished absorption of cholesterol. These changes are associated with liver fat content independent of body weight.

Genetic variation in PNPLA3 (adiponutrin) confers sensitivity to weight loss–induced decrease in liver fat in humans

BACKGROUND The rs738409 C→G single nucleotide polymorphism in the patatin-like phospholipase domain-containing 3 (PNPLA3; adiponutrin) leads to a missense mutation (I148M), which is associated with increased liver fat but not insulin resistance. The I148M mutation impedes triglyceride hydrolysis in vitro, and its carriers have an increased risk of developing severe liver disease. OBJECTIVE We explored whether the rs738409 PNPLA3 G allele influences the ability of weight loss to decrease liver fat or change insulin sensitivity. DESIGN We recruited 8 subjects who were homozygous for the rs738409 PNPLA3 G allele (PNPLA3-148MM) and 10 who were homozygous for the rs738409 PNPLA3 C allele (PNPLA3-148II). To allow comparison of changes in liver fat, the groups were matched with respect to baseline age, sex, body mass index, and liver fat. The subjects were placed on a hypocaloric low-carbohydrate diet for 6 d. Liver fat content (proton magnetic resonance spectroscopy), whole-body insulin sensitivity of glucose metabolism (euglycemic clamp technique), and lipolysis ([(2)H(5)]glycerol infusion) were measured before and after the diet. RESULTS At baseline, fasting serum insulin and C-peptide concentrations were significantly lower in the PNPLA3-148MM group than in the PNPLA3-148II group, as predicted by study design. Weight loss was not significantly different between groups (PNPLA3-148MM: -3.1 ± 0.5 kg; PNPLA3-148II: -3.1 ± 0.4 kg). Liver fat decreased by 45% in the PNPLA3-148MM group (P < 0.001) and by 18% in the PNPLA3-148II group (P < 0.01). CONCLUSION Weight loss is effective in decreasing liver fat in subjects who are homozygous for the rs738409 PNPLA3 G or C allele. This trial was registered at www.hus.fi as 233775.

Comparison of the Relative Contributions of Intra-Abdominal and Liver Fat to Components of the Metabolic Syndrome

Abdominally obese individuals with the metabolic syndrome often have excess fat deposition both intra‐abdominally (IA) and in the liver, but the relative contribution of these two deposits to variation in components of the metabolic syndrome remains unclear. We determined the mutually independent quantitative contributions of IA and liver fat to components of the syndrome, fasting serum (fS) insulin, and liver enzymes and measures of hepatic insulin sensitivity in 356 subjects (mean age 42 years, mean BMI 29.7 kg/m2) in whom liver fat and abdominal fat volumes were measured. IA and liver fat contents were correlated (r = 0.65, P < 0.0001). In multivariate linear regression analyses including either liver or IA fat, liver fat or IA fat explained variation in fS‐triglyceride (TG) and high‐density lipoprotein (HDL) cholesterol, plasma glucose, insulin and liver enzyme concentrations, and hepatic insulin sensitivity independent of age, gender, subcutaneous (SC) fat, and/or lean body mass (LBM). Including both liver and IA fat, liver and IA fat both explained variation in TG, HDL cholesterol, insulin and hepatic insulin sensitivity independent of each other and of age, gender, SC fat, and LBM. Liver fat independently predicted glucose and liver enzymes. SC fat and age explained variation in blood pressure. In conclusion, both IA and liver fat independently of each other explain variation in serum TG, HDL cholesterol, insulin concentrations and hepatic insulin sensitivity, thus supporting that both fat depots are important predictors of these components of the metabolic syndrome.

Adipose tissue inflammation and liver fat in patients with highly active antiretroviral therapy-associated lipodystrophy

In this cross-sectional study, we sought to determine whether gene expression of macrophage markers and inflammatory chemokines in lipoatrophic subcutaneous abdominal adipose tissue and liver fat content are increased and interrelated in human immunodeficiency virus (HIV)-1-positive, highly active antiretroviral therapy (HAART)-treated patients with lipodystrophy (HAART+LD+; n = 27) compared with those without (HAART+LD-; n = 13). The study groups were comparable with respect to age, gender, and body mass index. The HAART+LD+ group had twofold more intra-abdominal (P = 0.01) and 1.5-fold less subcutaneous (P = 0.091) fat than the HAART+LD- group. As we have reported previously, liver fat was 10-fold higher in the HAART+LD+ compared with the HAART+LD- group (P = 0.00003). Inflammatory gene expression was increased in HAART-lipodystrophy: CD68 4.5-fold (P = 0.000013), tumor necrosis factor (TNF)-alpha 2-fold (P = 0.0094), chemokine (C-C motif) ligand (CCL) 2 2.5-fold (P = 0.0024), CCL3 7-fold (P = 0.0000017), integrin alphaM (ITGAM) 3-fold (P = 0.00067), epidermal growth factor-like module containing, mucin-like, hormone receptor-like (EMR)1 2.5-fold (P = 0.0038), and a disintegrin and metalloproteinase domain (ADAM)8 3.5-fold (P = 0.00057) higher in the HAART+LD+ compared with the HAART+LD- group. mRNA concentration of CD68 (r = 0.37, P = 0.019), ITGAM (r = 0.35, P = 0.025), CCL2 (r = 0.39, P = 0.012), and CCL3 (r = 0.54, P = 0.0003) correlated with liver fat content. In conclusion, gene expression of markers of macrophage infiltration and adipose tissue inflammation is increased in lipoatrophic subcutaneous abdominal adipose tissue of patients with HAART-associated lipodystrophy compared with those without. CD68, ITGAM, CCL2, and CCL3 expression is significantly associated with accumulation of liver fat.

Gene Expression and Immunohistochemistry in Adipose Tissue of HIV Type 1–Infected Patients with Nucleoside Analogue Reverse-Transcriptase Inhibitor–Associated Lipoatrophy

BACKGROUND Long-term use of both zidovudine (AZT) and stavudine (d4T) is associated with lipoatrophy, but it occurs possibly through different mechanisms. METHODS Surgical biopsy specimens of subcutaneous adipose tissue were obtained from 18 human immunodeficiency virus type 1 (HIV-1)-infected lipoatrophic patients (the LA+ group) who were treated with either zidovudine (the AZT+LA+ group; n = 10) or stavudine (the d4T+LA+ group; n = 8) and from 10 nonlipoatrophic HIV-1-infected patients (the LA- group) who received antiretroviral therapy. Mitochondrial DNA (mtDNA) copy numbers, gene expression, and immunohistochemistry data were analyzed. RESULTS mtDNA copy numbers were significantly reduced in the LA+ group, compared with the LA- group, and in the d4T+LA+ group, compared with the AZT+LA+ group. The ratio of mtDNA-encoded cytochrome COX3 to nuclear DNA-encoded COX4 expression was significantly lower in the LA+ group than in the LA- group. Compared with the LA- group, the LA+ group had significantly lower expression of genes involved in adipogenesis (SREBP1c and CEBPB), lipid (fatty acid synthase), and glucose (GLUT4) metabolism. Expression of genes involved in mitochondrial biogenesis (PGC1B), apoptosis (FAS), inflammation (IL1B), oxidative stress (PCNA and SOD1), and lamin B was significantly higher in the LA+ group than in the LA- group. The d4T+LA+ group had significantly lower expression of genes involved in mitochondrial biogenesis (POLG1), energy metabolism (the COX3/COX4 ratio), adipogenesis (SREBP1c and CEBPA), perilipin, and hexokinase than did the AZT+LA+ group. There were 7-fold more macrophages in adipose tissue specimens obtained from patients in the LA+ group, compared with the LA- group. CONCLUSIONS Lipoatrophy is characterized by mtDNA depletion, inflammation, and signs of apoptosis. Changes were more profound in the d4T+LA+ group than in the AZT+LA+ group.

Nonalcoholic Fatty Liver Disease: Detection of Elevated Nicotinamide Adenine Dinucleotide Phosphate with in Vivo 3.0-T 31P MR Spectroscopy with Proton Decoupling

PURPOSE To determine if 3.0-T proton-decoupled phosphorus 31 ((31)P) magnetic resonance (MR) spectroscopy can be used to differentiate between stages of nonalcoholic fatty liver disease (NAFLD) by resolving the components of phosphomonoester (PME) and phosphodiester (PDE) and enabling detection of a greater number of other phosphorus-containing compounds. MATERIALS AND METHODS This study was approved by the ethics committee of Helsinki University Central Hospital, and written informed consent was obtained from all study subjects. A 3.0-T clinical imager was used to obtain proton-decoupled (31)P MR spectra in the liver of control subjects (n = 12), patients with biopsy-proved simple steatosis due to nonalcoholic causes (nonalcoholic fatty liver, n = 13; nonalcoholic steatohepatitis [NASH], n = 9), and patients with cirrhosis (n = 9) to determine PME, phosphoethanolamine (PE), phosphocholine, PDE, glycerophosphocholine (GPC), glycerophosphoryl ethanolamine, uridine diphosphoglucose, nicotinamide adenine dinucleotide phosphate (NADPH), inorganic phosphate, phosphoenolpyruvate, and alpha-, beta- and gamma-nucleotide triphosphate levels. Liver fat was determined with hydrogen 1 MR spectroscopy. Differences between the disease groups were analyzed with one-way analysis of variance. RESULTS The PME/(PME + PDE), PME/PDE, and PE/(PME + PDE) ratios were higher and the GPC/(PME + PDE) ratio was lower in patients with cirrhosis than in the other study groups (P < or = .001, one-way analysis of variance). The NADPH/(PME + PDE) ratio was higher in patients with NASH and those with cirrhosis than in control subjects (P < .05, post hoc analyses) and correlated with disease severity (P = .007). CONCLUSION NADPH, a marker of inflammation and fibrinogenic activity in the liver, is increased in patients with NASH and those with cirrhosis. Proton-decoupled (31)P 3.0-T MR spectroscopy shows promise in the differentiation of NAFLD stages.

Increased coagulation factor VIII, IX, XI and XII activities in non-alcoholic fatty liver disease

Background and aims: Obesity and the metabolic syndrome are established risk factors of venous thromboembolism. As most coagulation factors are produced exclusively by the liver and non‐alcoholic fatty liver disease (NAFLD) is tightly related to metabolic disorders, we aimed at studying the association of liver fat with various coagulation factor activities.

Genetic variation in PNPLA3 but not APOC3 influences liver fat in non-alcoholic fatty liver disease

Background and Aim:  A recent study in Indian subjects suggested common variants in apolipoprotein C3 (APOC3) (T‐455C at rs2854116 and C‐482T at rs2854117) to contribute to non‐alcoholic fatty liver disease (NAFLD), plasma apoC3 and triglyceride concentrations. Our aim was to determine the contribution of genetic variation in APOC3 on liver fat content and plasma triglyceride and apoC3 concentrations in a larger European cohort.