Non alcoholic fatty liver disease and risk of incident diabetes in subjects who are not obese

Background and aims

It is not known whether non alcoholic fatty liver disease (NAFLD) is a risk factor for diabetes in non obese, non centrally-obese subjects. Our aim was to investigate relationships between fatty liver, insulin resistance and a biomarker score for liver fibrosis with incident diabetes at follow up, in subjects who were neither obese nor centrally-obese.

Smoking is associated with severity of liver fibrosis but not with histological severity in nonalcoholic fatty liver disease. Results from a cross-sectional study

Objectives: To assess the influence of smoking on histological disease severity and fibrosis in real-world NAFLD patients.

Material and methods: Consecutive NAFLD patients were identified with liver biopsies performed between 2008 and 2015. Characteristics such as smoking status and total number of pack years were collected. Biopsies were revised and BRUNT fibrosis and NAFLD activity score (NAS) determined. Patients with a high NAS (≥5) were compared to patients with a low NAS (<5) and with advanced fibrosis (stage 3–4) to patients with no-early fibrosis (stage 0–2). Patients with a history of smoking (current or past smoker) were defined ever smokers.

Results: Fifty-six patients were included (mean age 49?±?14.3, 68.9% males and 39.3% history of smoking). Ever smokers had a higher fibrosis score than never smokers; two (IQR 0–3) versus one (IQR 1–1.5) (p?=?.040). Patients with advanced fibrosis smoked significantly more pack years than patients with no-early fibrosis; 10.6 (IQR 0–25.8) versus 0 (IQR 0–7) (p?=?.011). There is a weak to moderate correlation between fibrosis stage and number of pack years (Spearman’s Rho?=?0.341, p?=?.012). There was no difference in NAS between never and ever smokers; 2.8?±?1.5 versus 3.3?±?1.4 (p?=?.205). Patients with NAS <5 had a median number of pack years of 0 (IQR 0–9) versus a median of 10.3 pack years (IQR 0–24) in patients with NAS ≥5 (p?=?.127).

Conclusion: Smoking is associated with severity of NAFLD-related liver fibrosis but not with histological disease severity. This supports the recommendation to cease smoking for NAFLD patients.     

Expression of adiponectin and its receptors in livers of morbidly obese patients with non-alcoholic fatty liver disease

Background and Aim:  Obesity is one of the risk factors for non-alcoholic fatty liver disease (NAFLD) and a common disease that comprises simple steatosis and non-alcoholic steatohepatitis (NASH), and can eventually lead to liver cirrhosis. Adiponectin is an adipocyte-derived protein that has anti-obesity, antidiabetic and anti-inflammatory properties, and is considered to possess a hepatoprotective function. Its role in the development and progression of NAFLD in morbidly obese patients is unknown. In this study, we examined the expression levels of adiponectin and its receptors in liver biopsies of morbidly obese patients and then determined whether there was an association with the disease severity.
Methods:  Liver biopsies from 30 morbidly obese patients (18 NASH vs 12 steatosis) were analyzed. The needle core biopsies were subjected to routine histological examination and stained immunohistochemically for adiponectin, adiponectin receptor I (adipoRI) and receptor II (adipoRII).
Results:  The two groups were comparable with respect to body mass index, age and gender distribution. The expression of adiponectin decreased in liver biopsies with NASH as compared to those with simple steatosis (1.61 ± 0.70 vs 2.25 ± 0.75, P  = 0.028). Spearman's rank correlation coefficient analysis showed that the staining intensity of adiponectin negatively correlated with the grade of inflammation ( r  = −0.368, P  = 0.045) and stage of fibrosis ( r  = −0.380, P  = 0.038). There was no significant difference in expression of adipoRI and adipoRII between the two groups.
Conclusion:  These findings indicate that decreased liver adiponectin expression may play a role in the development and progression of NAFLD, from simple steatosis to NASH, in morbidly obese patients.     

Serum ferritin is a discriminant marker for both fibrosis and inflammation in histologically proven non‐alcoholic fatty liver disease patients

Introduction: Differentiation between steatosis and non‐alcoholic steatohepatitis (NASH) in non‐alcoholic fatty liver disease (NAFLD) is important as NASH progress to cirrhosis. No specific laboratory/imaging technique exists either to diagnose NASH or to select patients for liver biopsy. Patients and methods: We evaluated serum ferritin and the features of metabolic syndrome with respect to histological inflammation and/or fibrosis in NAFLD patients. The Kleiner scoring system was used to classify NAFLD in consecutive liver biopsies. One hundred and eleven patients: median age 52.6, 64 males, obesity 62, diabetes mellitus (DM) 58, arterial hypertension 26 and hyperlipidaemia 40%. Results: Histologically, 40.7 had fatty liver, 30.6% had borderline NASH, 28.7% had NASH and 11% had cirrhosis. Multivariate regression showed that diabetes, serum ferritin concentrations, body mass index (BMI) and AST were independently associated with NASH: together, the areas under the receiver operating characteristic (AUROC) was 0.91 (95% confidence interval 0.86–0.96); fibrosis was associated with ferritin concentrations and BMI: AUROC 0.87, portal inflammation with ferritin and DM: AUROC 0.82, while lobular inflammation was associated with BMI, DM and ferritin: AUROC 0.85. Conclusion: Serum ferritin concentrations and BMI are strongly associated with fibrosis, portal and lobular inflammation in NAFLD patients. Both ferritin and BMI are potential discriminant markers to select patients for liver biopsy and are associated with inflammation and fibrosis.     

Visualized macrophage dynamics and significance of S100A8 in obese fat

Chronic low-grade inflammation of adipose tissue plays a crucial role in the pathophysiology of obesity. Immunohistological microscopic analysis in obese fat tissue has demonstrated the infiltration of several immune cells such as macrophages, but dynamics of immune cells have not been fully elucidated and clarified. Here, by using intravital multiphoton imaging technique, to our knowledge for the first time, we analyzed and visualized the inflammatory processes in adipose tissue under high-fat and high-sucrose (HF/HS) diet with lysozyme M-EGFP transgenic (LysM^EGFP) mice whose EGFP was specifically expressed in the myelomonocytic lineage. Mobility of LysM^EGFP-positive macrophages was shown to be activated just 5 d after HF/HS diet, when the distinct hypertrophy of adipocytes and the accumulation of macrophages still have not become prominent. Significant increase of S100A8 was detected in mature adipocyte fraction just 5 d after HF/HS diet. Recombinant S100A8 protein stimulated chemotactic migration in vitro and in vivo, as well as induced proinflammatory molecules, both macrophages and adipocytes, such as TNF-α and chemokine (C-C motif) ligand 2. Finally, an antibody against S100A8 efficiently suppressed the HF/HS diet-induced initial inflammatory change, i.e., increased mobilization of adipose LysM^EGFP-positive macrophages, and ameliorated HF/HS diet-induced insulin resistance. In conclusion, time-lapse intravital multiphoton imaging of adipose tissues identified the very early event exhibiting increased mobility of macrophages, which may be triggered by increased expression of adipose S100A8 and results in progression of chronic inflammation in situ.Obesity, especially visceral fat obesity, is a central player in the development of metabolic syndrome and in its clinical consequences (1–4). Chronic low-grade inflammation of adipose tissue has been demonstrated to be critical in the pathogenesis of obesity (5, 6). In terms of innate immune cells, the defining feature of adipose tissue inflammation in obesity is a marked increase of macrophage infiltration into adipose tissue (6, 7). The macrophages in inflamed adipose tissue are predominantly inflammatory M1 macrophages producing proinflammatory cytokines such as TNF-α (8). The other immune cells, such as T/B lymphocytes (9, 10), neutrophils (11), and eosinophils (12), have also been shown to play significant roles in adipose tissue inflammation and consequent metabolic disorders. However, most of these observations are based on immunohistological and flow cytometric analyses. The dynamic nature of immune cells has not been fully elucidated in adipose tissue during the progression of obesity. Adipose tissue is considered to be a key site of interaction between adipocytes and other immune system effectors, which may highlight the necessity to analyze immune cell dynamics in obese adipose tissue in vivo.Chemokines and other inflammatory mediators have been proposed to be involved in adipose tissue inflammation in obesity. Several potential targets of modulation of inflammatory response have been demonstrated. Among them, there is considerable evidence for the pathophysiological role of the chemokine (C-C motif) ligand 2 [CCL2; monocyte chemoattractant protein-1 (MCP-1)]/chemokine (C-C motif) receptor 2 (CCR2) pathway in monocyte/macrophage infiltration into obese adipose tissues (13, 14). In addition, danger-associated molecular patterns, also called alarmins, which are released from damaged tissues and stressed cells, have caused sterile immune responses such as obesity-induced chronic inflammation. Pattern-recognition receptors such as Toll-like receptors (TLRs) and NOD-like receptors are involved in binding and responding to alarmins, including high-mobility group box chromosomal protein 1 (HMGB1) (15), S100 proteins (16, 17), saturated fatty acid (18, 19), oxidized LDL (20), and degradation products of extracellular matrices (21). Furthermore, adipose-derived saturated fatty acids have been shown to directly activate TLR4 and TLR2, not only of macrophages but also of adipocytes themselves, as endogenous ligands, resulting in the production of proinflammatory cytokines and chemokines and the dysregulation of adipocytokines (18, 19).Chronic inflammation is a complex phenomenon, and multiple factors have been shown to be intricately involved in the progression of adipose tissue inflammation when it has happened. However, the early events triggering the chronic inflammatory cascades in adipose tissues have not been understood. Here, by means of intravital multiphoton microscopy of adipose tissues, we visualized time-dependent changes of immune cell dynamics during the development of obesity, and succeeded in identifying the early triggering event of chronic inflammation in obese adipose tissue.     

Nonalcoholic fatty liver disease in severely obese subjects

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) has been consistently associated with obesity and insulin resistance. Nonalcoholic steatohepatitis (NASH) is a histological entity within NAFLD that can progress to cirrhosis. The exact prevalence of NASH in severe obesity is unknown. It is unclear whether differences in insulin sensitivity exist among subjects with NASH and simple fatty liver. OBJECTIVE: To evaluate the prevalence and correlates of NASH and liver fibrosis in a racially diverse cohort of severely obese subjects. DESIGN: Ninety-seven subjects were enrolled. Liver biopsies, indirect markers of insulin resistance, metabolic parameters, and liver function tests were obtained. RESULTS: Thirty-six percent of subjects had NASH and 25% had fibrosis. No cirrhosis was diagnosed on histology. Markers of hyperglycemia, insulin resistance, and the metabolic syndrome but not body mass index were associated with the presence of NASH and fibrosis. Elevated transaminase levels correlated strongly with NASH and fibrosis but 46% subjects with NASH had normal transaminases. Subjects with NASH had more severe insulin resistance when compared to those with simple fatty liver. A signal detection model incorporating AST and the presence of diabetes predicted the presence of NASH while another incorporating ALT and HbA1C predicted the presence of fibrosis. CONCLUSIONS: NAFLD is associated with the metabolic syndrome rather than excess adipose tissue in severe obesity. Insulin resistance is higher in subjects with NASH versus those with simple fatty liver. Statistical models incorporating markers of liver injury and hyperglycemia may be useful in predicting the presence of liver pathology in this population.     

Subclinical and clinical atherosclerosis in non-alcoholic fatty liver disease is associated with the presence of hypertension

Background and aimsNon-alcoholic fatty liver disease (NAFLD) is associated with increased cardiovascular (CV) risk. However, it is unclear whether NAFLD contributes independently to the development of CV disease. Our study aimed at assessing the differences in several indices of atherosclerosis, arterial stiffness and cardiac morphology among patients with isolated NAFLD, isolated hypertension (HT) or a combination of the two conditions.Methods and resultsA total of 169 participants (mean age = 50.4 ± 10.2 yrs; males = 73.6%) were divided according to the presence of NAFLD and HT into three groups: only NAFLD (55 patients), only HT (49 patients), and NAFLD + HT (65 patients). Exclusion criteria were a BMI≥35 kg/m² and a diagnosis of diabetes mellitus. Carotid ultrasonography was performed to measure markers of atherosclerosis and arterial stiffness. Cardiac remodeling was analyzed using echocardiography. The prevalence of subclinical and overt atherosclerosis was significantly higher in the NAFLD + HT patients as compared to the other two groups (atherosclerotic plaques: 43.1%, 10.9%, and 22.4% (p < 0.001) in NAFLD + HT, NAFLD, and HT groups, respectively). No differences were found among indices of arterial stiffening and cardiac remodeling across the three groups. In multivariate regression analysis, the coexistence of NAFLD and HT was an independent risk factor for overt atherosclerosis (OR = 4.88, CI 95% 1.14–20.93), while no association was found when either NAFLD or HT was considered alone.ConclusionOvert atherosclerosis was significantly present only in NAFLD + HT patients, but not in patients with isolated NAFLD. This implies that the impact of NAFLD on vascular structure and function could depend on the coexistence of other major CV risk factors, such as HT.     

In humans the adiponectin receptor R2 is expressed predominantly in adipose tissue and linked to the adipose tissue expression of MMIF‐1

In this study, the regional adipose tissue‐adiponectin (AT‐ADN) and adiponectin receptor (R1 and R2) expression and their relation with metabolic parameters, circulating and AT‐derived cytokine expressions were compared. Paired subcutaneous adipose tissue (SCAT) and visceral adipose tissue (VAT) were taken from 18 lean and 39 obese humans, AT‐mRNA expression of adipokines analysed by RT‐PCR and corresponding serum levels by enzyme‐linked immunosorbent assay (ELISA). R1 and R2 adipocyte expression was compared with 17 other human tissues. ADN‐gene expression was lower in VAT than SCAT [mean (SD) 1.54 (1.1) vs. 2.84 (0.87); p < 0.001], and lower in obese subjects (VAT : p = 0.01;SCAT : p < 0.001). SCAT‐ADN correlated positively with serum ADN (r = 0.33;p = 0.036) but not VAT‐ADN. AT expressions of ADN and macrophage migration inhibiting factor (MMIF), IL18 and cluster of differentiation factor 14 (CD14) in both depots showed inverse correlations. R1 and R2 were expressed ubiquitously and R2 highest in SCAT, and this is much higher (×100) than R1 (×100). R expression was similar in lean and obese subjects and unrelated to the metabolic syndrome, however, receptors correlated with VAT‐MMIF (R 1: r = 0.4;p = 0.008;R 2: r = 0.35,p = 0.02) and SCAT‐MMIF expression (R 2: r = 0.43;p = 0.004). Unlike ADN, its receptors are expressed in many human tissues. Human R2 expression is not highest in the liver but in AT where it is associated with MMIF expression. The adiponectin‐dependent insulin‐sensitizing action of thiazolidinediones is thus probably to differ amongst species with weaker effects on the human liver.     

Blockade of kinin B(1) receptor reverses plasma fatty acids composition changes and body and tissue fat gain in a rat model of insulin resistance

Aim: Kinin B₁ receptor (B₁R) contributes to insulin resistance through a mechanism involving oxidative stress. This study examined the effect of B₁R blockade on the changes in plasma fatty acids composition, body and tissue fat mass and adipose tissue inflammation that influence insulin resistance. Methods: Sprague–Dawley rats were fed with 10% D‐glucose or tap water (Control) for 13 weeks and during the last week, rats were administered the B₁R antagonist SSR240612 (10 mg/kg/day, gavage) or vehicle. The following parameters were assessed: plasma fatty acids (by gas chromatography), body composition (by EchoMRI), metabolic hormone levels (by radioimmunoassay), expression of B₁R and inflammatory markers in adipose tissue (by Western blot and qRT‐PCR). Results: Glucose feeding significantly increased plasma levels of glucose, insulin, leptin, palmitoleic acid (16:1n‐7), oleic acid (18:1n‐9), Δ6 and Δ9 desaturases while linoleic acid (18:2n‐6), arachidonic acid (20:4n‐6) and Δ5 desaturase were decreased. SSR240612 reduced plasma levels of insulin, glucose, the homeostasis model assessment index of insulin resistance, palmitoleic acid and n‐7 family. Alterations of Δ5, Δ6 and Δ9 desaturases were normalized by SSR240612. The B₁R antagonist also reversed the enhancing effect of glucose feeding on whole body and epididymal fat mass and on the expression of macrophage CD68, interleukin‐1β, tumour necrosis factor‐α and inducible nitric oxide synthase in retroperitoneal adipose tissue. B₁R protein and mRNA were not detected in retroperitoneal adipose tissue. Conclusion: Insulin resistance in glucose‐fed rats is associated with low state inflammation in adipose tissue and plasma fatty acids changes which are reversed by B₁R blockade. These beneficial effects may contribute to insulin sensitivity improvement and the prevention of obesity.     

Genetic determinants of susceptibility and severity in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) in most patients involves only simple hepatic steatosis; however, a minority develop progressive steatohepatitis. Family studies and inter-ethnic differences in susceptibility suggest that genetic factors may be important risk determinants for progressive disease. Polymorphisms in genes affecting lipid metabolism, cytokines, fibrotic mediators and oxidative stress may be associated with steatohepatitis and/or fibrosis, but most of these findings require replication. A recent finding that a nonsynonymous polymorphism in the PNPLA3 gene predicts the extent of steatosis in NAFLD has been replicated in at least eight studies, with several studies also demonstrating an association with fibrosis. A new genome-wide association study has identified several additional novel associations with NAFLD severity. Other disease genes may be identified by similar approaches in the future.