Association of nonalcoholic fatty liver disease with a single nucleotide polymorphism on the gene encoding leptin receptor

Leptin (Lep), a 16-kDa polypeptide hormone, exerts its action through the leptin receptor (LepRb), a member of the class I cytokine receptor family. Both leptin and LepRb probably have been implicated in pathogenesis of nonalcoholic fatty liver disease (NAFLD). This study was designed to assess the role of soluble leptin and LepRb in NAFLD and to investigate whether leptin receptor gene (LepR) single nucleotide polymorphism (SNP; ID rs6700896) influences NAFLD complicated with or without type 2 diabetes mellitus (T2DM). Blood samples from 90 obese NAFLD cases and 30 lean controls of matched age and sex were recruited in the study. Among the NAFLD patients, 32 were T2DM. Plasma leptin and LepRb levels were measured by enzyme linked immunoassay (ELISA). Lipids profile, glucose metabolic parameters, and insulin concentration were measured for all participants. Body mass index (BMI) and insulin resistance (IR) were calculated as well. Genotyping was done using SNP (rs6700986) for LepR gene. Significant difference was reported between NAFLD with or without T2DM and control regarding biochemical markers and LepR genotype and allele frequencies. Mutant homozygous and heterozygous LepR genotype and mutant allele were significantly higher in mild-severe steatosis and in NAFLD with T2DM when compared with mild steatosis and those without T2DM. Frequencies of mutant LepR polymorphism were significantly associated with IR increment. Elevated leptin level seems to be a feature of steatosis, and it appears to increase as hepatocyte steatosis develops. Moreover, polymorphism of LepR gene contributes to the onset of NAFLD by regulating lipid metabolism and affecting insulin sensitivity.     

Fatty acids and the endoplasmic reticulum in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) represents a burgeoning public health concern in westernized nations. The obesity-related disorder is associated with an increased risk of cardiovascular disease, type 2 diabetes and liver failure. Although the underlying pathogenesis of NAFLD is unclear, increasing evidence suggests that excess saturated fatty acids presented to or stored within the liver may play a role in both the development and progression of the disorder. A putative mechanism linking saturated fatty acids to NAFLD may be endoplasmic reticulum (ER) stress. Specifically, excess saturated fatty acids may induce an ER stress response that, if left unabated, can activate stress signaling pathways, cause hepatocyte cell death, and eventually lead to liver dysfunction. In the current review we discuss the involvement of saturated fatty acids in the pathogenesis of NAFLD with particular emphasis on the role of ER stress.     

n-3 PUFA and lipotoxicity

Excess lipid accumulation in nonadipose tissues may occur in the setting of high levels of plasma free fatty acids or triglycerides (TGs) in a process called “lipotoxicity”. Evidence from human studies and animal models suggests that lipid accumulation in the heart, skeletal muscle, pancreas, and liver play an important role in the pathogenesis of heart failure, obesity, metabolic syndrome, and type 2 diabetes mellitus (T2DM). During the past few years, several studies have shown that n-3 polyunsaturated fatty acids (PUFA) have potentially cardioprotective effects, especially in high-risk patients with dyslipidemia, and might therefore be expected to be of benefit in T2DM. Moreover, new information has demonstrated the beneficial effects of consuming n-3 PUFA in preventing the complications of lipotoxicity. n-3 PUFA dietary intake thus had positive effects on fatty liver in patients with non-alcoholic fatty liver disease (NAFLD), with an improvement in liver echotexture and a significant regression of hepatic brightness, associated with improved liver hemodynamics. The n-3 PUFA also had beneficial effects on ectopic fat accumulation inside the heart, with stabilization of cardiac myocytes and antiarrhythmic effects. On the other hand, recent data from animal models suggest that oral dosing of eicosapentaenoic acid (EPA) could contribute to protect against β-cell lipotoxicity. This review discusses the latest hypotheses regarding lipotoxicity, concentrating on the impact of the n-3 PUFA that contribute to ectopic lipid storage, affecting organ function. Further human studies are needed to test the evidence and elucidate the mechanisms involved in this process.     

Insulin resistance and oxidative stress interdependency in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is emerging as a major cause of chronic liver disease in association with the rising prevalence of obesity and type 2 diabetes in the population. Oxidative stress and insulin resistance (IR) are major contributors in the pathogenesis of NAFLD and in the progression from steatosis to steatohepatitis. Recently, Houstis and colleagues reported that reactive oxygen species have a causal role in multiple forms of IR, a phenomenon that can further promote exacerbation of oxidative stress. The improvement of the knowledge of these interrelationships should contribute to elucidate pathogenic pathways and design effective treatments for NAFLD.     

MicroRNAs in the Pathogenesis of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD), or, more accurately, metabolic associated fatty liver disease, accounts for a large proportion of chronic liver disorders worldwide and is closely associated with other conditions such as cardiovascular disease, obesity, and type 2 diabetes mellitus. NAFLD ranges from simple steatosis to nonalcoholic steatohepatitis (NASH) and can progress to cirrhosis and, eventually, also hepatocellular carcinoma. The morbidity and mortality associated with NAFLD are increasing rapidly year on year. Consequently, there is an urgent need to understand the etiology and pathogenesis of NAFLD and identify effective therapeutic targets. MicroRNAs (miRNAs), important epigenetic factors, have recently been proposed to participate in NAFLD pathogenesis. Here, we review the roles of miRNAs in lipid metabolism, inflammation, apoptosis, fibrosis, hepatic stellate cell activation, insulin resistance, and oxidative stress, key factors that contribute to the occurrence and progression of NAFLD. Additionally, we summarize the role of miRNA-enriched extracellular vesicles in NAFLD. These miRNAs may comprise suitable therapeutic targets for the treatment of this condition.     

Circulating Level of CTRP1 in Patients with Nonalcoholic Fatty Liver Disease (NAFLD): Is It through Insulin Resistance?

Nonalcoholic fatty liver disease (NAFLD) is considered as one of the most common liver diseases. It is robustly linked to obesity and insulin resistance and is regarded as hepatic manifestation of metabolic syndrome (MetS). Adipokines are involved in the pathophysiology of liver diseases. The aim of this study was to evaluate the plasma concentrations of CTRP1 (complement-C1q TNF-related protein 1) in 22 patients with NAFLD, 22 patients with type 2 diabetes mellitus (T2DM), 22 patients with NAFLD+T2DM and 21 healthy controls, as well as their correlation with the level of metabolic and hepatic parameters. Plasma concentration of CTRP1 was measured with ELISA method. Plasma concentration of CTRP1 in patients with NAFLD, T2DM and NAFLD+T2DM were significantly higher than healthy subjects (p<0.0001). Moreover, we observed significant positive correlations between plasma level of CTRP1 and fasting blood glucose (FBG) (p<0.001), homeostasis model assessment of insulin resistance (HOMA-IR) (p<0.001), body mass index (BMI) (p = 0.001), alanine amino transferase (ALT) (p = 0.002), gamma glutamyl transferase (γ-GT) (p<0.001) and liver stiffness (LS) (p<0.001). Our results indicate the strong association of CTRP1 with insulin resistance in NAFLD. Also, it seems that CTRP1 can be considered as an emerging biomarker for NAFLD, however, more studies are necessary to unravel the role of CTRP1 in NAFLD pathogenesis.     

A Model of Metabolic Syndrome and Related Diseases with Intestinal Endotoxemia in Rats Fed a High Fat and High Sucrose Diet

Aim

We sought develop and characterize a diet-induced model of metabolic syndrome and its related diseases.

Methods

The experimental animals (Spague-Dawley rats) were randomly divided into two groups, and each group was fed a different feed for 48 weeks as follows: 1) standard control diet (SC), and 2) a high sucrose and high fat diet (HSHF). The blood, small intestine, liver, pancreas, and adipose tissues were sampled for analysis and characterization.

Results

Typical metabolic syndrome (MS), non-alcoholic fatty liver disease (NAFLD), and type II diabetes (T2DM) were common in the HSHF group after a 48 week feeding period. The rats fed HSHF exhibited signs of obesity, dyslipidemia, hyperglycaemia, glucose intolerance, and insulin resistance (IR). At the same time, these animals had significantly increased levels of circulating LPS, TNFα, and IL-6 and increased ALP in their intestinal tissue homogenates. These animals also showed a significant reduction in the expression of occluding protein. The HSHF rats showed fatty degeneration, inflammation, fibrosis, cirrhosis, and lipid accumulation when their liver pathologies were examined. The HSHF rats also displayed increased islet diameters from 12 to 24 weeks, while reduced islet diameters occurred from 36 to 48 weeks with inflammatory cell infiltration and islet fat deposition. The morphometry of adipocytes in HSHF rats showed hypertrophy and inflammatory cell infiltration. HSHF CD68 analysis showed macrophage infiltration and significant increases in fat and pancreas size. HSHF Tunel analysis showed significant increases in liver and pancreas cell apoptosis.

Conclusions

This work demonstrated the following: 1) a characteristic rat model of metabolic syndrome (MS) can be induced by a high sucrose and high fat diet, 2) this model can be used to research metabolic syndrome and its related diseases, such as NAFLD and T2DM, and 3) intestinal endotoxemia (IETM) may play an important role in the pathogenesis of MS and related diseases, such as NAFLD and T2DM.     

Hepatic deficiency of selenoprotein S exacerbates hepatic steatosis and insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is closely associated with insulin resistance (IR) and type 2 diabetes mellitus (T2DM), which are all complex metabolic disorders. Selenoprotein S (SelS) is an endoplasmic reticulum (ER) resident selenoprotein involved in regulating ER stress and has been found to participate in the occurrence and development of IR and T2DM. However, the potential role and mechanism of SelS in NAFLD remains unclear. Here, we analyzed SelS expression in the liver of high-fat diet (HFD)-fed mice and obese T2DM model (db/db) mice and generated hepatocyte-specific SelS knockout (SelS^H-KO) mice using the Cre-loxP system. We showed that hepatic SelS expression levels were significantly downregulated in HFD-fed mice and db/db mice. Hepatic SelS deficiency markedly increased ER stress markers in the liver and caused hepatic steatosis via increased fatty acid uptake and reduced fatty acid oxidation. Impaired insulin signaling was detected in the liver of SelS^H-KO mice with decreased phosphorylation levels of insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/Akt), which ultimately led to disturbed glucose homeostasis. Meanwhile, our results showed hepatic protein kinase Cɛ (PKCɛ) activation participated in the negative regulation of insulin signaling in SelS^H-KO mice. Moreover, the inhibitory effect of SelS on hepatic steatosis and IR was confirmed by SelS overexpression in primary hepatocytes in vitro. Thus, we conclude that hepatic SelS plays a key role in regulating hepatic lipid accumulation and insulin action, suggesting that SelS may be a potential intervention target for the prevention and treatment of NAFLD and T2DM.Subject terms: Metabolic syndrome, Obesity     

Association between resistin + 299A/A genotype and nonalcoholic fatty liver disease in Chinese patients with type 2 diabetes mellitus

Objective

To investigate the relationship between the resistin intronic + 299G/A polymorphism and nonalcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM).

Methods

We selected 738 T2DM patients, including 395 with NAFLD and 343 without fatty liver disease, as well as 279 healthy control individuals, and analyzed their resistin + 299G/A polymorphism genotype by polymerase chain reaction–restriction fragment length polymorphism.

Results

Plasma resistin levels in T2DM patients with NAFLD were at the highest (P < 0.05). The frequency of AA genotype at the + 299 site of the resistin gene in patients with concurrent T2DM combined with NAFLD was significantly different from that in the control (P < 0.05). The AA genotype was found to be associated with a 1.80-fold increased risk for T2DM combined with NAFLD, 2.05-fold increased risk for obesity and 2.37-fold increased risk for obesity of abdominal type compared to the GG (P < 0.05, respectively). The multivariate non-conditional logistic regression model analysis further shows that the AA genotype is a risk factor for the development of NAFLD in T2DM patients (OR, 2.32; 95% CI, 1.05–4.68; P < 0.05).

Conclusion

The resistin + 299AA genotype may be associated with increases in the risk of the NAFLD development in T2DM patients.     

Long-term fatty liver-induced insulin resistance in orotic acid-induced nonalcoholic fatty liver rats

We investigated whether fatty liver preceded insulin resistance or vice versa using a long-term orotic acid (OA)-induced nonalcoholic fatty liver disease (NAFLD) model without the confounding effects of obesity and hyperlipidemia and explored the role of the liver in insulin resistance. Male Wistar rats were fed with or without OA supplementation for 30, 60, and 90 days. The NAFLD group showed increased liver lipid at 30, 60, and 90 days; glucose intolerance was noted at 60 and 90 days. Furthermore, partial liver proteins and gene expressions related to upstream signaling of insulin were decreased. However, the liver glycogen content was elevated, and gluconeogenesis genes expressions were obviously decreased at 90 days. The occurrence of fatty liver preceded insulin resistance in OA-induced NAFLD without the interference of obesity and hyperlipidemia, and hepatic insulin resistance may not play a conclusive role in insulin resistance in this model.     

Genetic determinants of hepatic steatosis in man

Hepatic steatosis is one of the most common liver disorders in the general population. The main cause of hepatic steatosis is nonalcoholic fatty liver disease (NAFLD), representing the hepatic component of the metabolic syndrome, which is characterized by type 2 diabetes, obesity, and dyslipidemia. Insulin resistance and excess adiposity are considered to play key roles in the pathogenesis of NAFLD. Although the risk factors for NAFLD are well established, the genetic basis of hepatic steatosis is largely unknown. Here we review recent progress on genomic variants and their association with hepatic steatosis and discuss the potential impact of these genetic studies on clinical practice. Identifying the genetic determinants of hepatic steatosis will lead to a better understanding of the pathogenesis and progression of NAFLD.     

The role of fructose-enriched diets in mechanisms of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) currently affects 20%-30% of adults and 10% of children in industrialized countries, and its prevalence is increasing worldwide. Although NAFLD is a benign form of liver dysfunction, it can proceed to a more serious condition, nonalcoholic steatohepatitis (NASH), which may lead to liver cirrhosis and hepatocellular carcinoma. NAFLD is accompanied by obesity, metabolic syndrome and diabetes mellitus, and evidence suggests that fructose, a major caloric sweetener in the diet, plays a significant role in its pathogenesis. Inflammatory progression to NASH is proposed to occur by a two-hit process. The first "hit" is hepatic fat accumulation owing to increased hepatic de novo lipogenesis, inhibition of fatty acid beta oxidation, impaired triglyceride clearance and decreased very-low-density lipoprotein export. The mechanisms of the second "hit" are still largely unknown, but recent studies suggest several possibilities, including inflammation caused by oxidative stress associated with lipid peroxidation, cytokine activation, nitric oxide and reactive oxygen species, and endogenous toxins of fructose metabolites.     

Mice Lacking Natural Killer T Cells Are More Susceptible to Metabolic Alterations following High Fat Diet Feeding

Current estimates suggest that over one-third of the adult population has metabolic syndrome and three-fourths of the obese population has non-alcoholic fatty liver disease (NAFLD). Inflammation in metabolic tissues has emerged as a universal feature of obesity and its co-morbidities, including NAFLD. Natural Killer T (NKT) cells are a subset of innate immune cells that abundantly reside within the liver and are readily activated by lipid antigens. There is general consensus that NKT cells are pivotal regulators of inflammation; however, disagreement exists as to whether NKT cells exert pathogenic or suppressive functions in obesity. Here we demonstrate that CD1d^−/− mice, which lack NKT cells, were more susceptible to weight gain and fatty liver following high fat diet (HFD) feeding. Compared with their WT counterparts, CD1d^−/− mice displayed increased adiposity and greater induction of inflammatory genes in the liver suggestive of the precursors of NAFLD. Calorimetry studies revealed a significant increase in food intake and trends toward decreased metabolic rate and activity in CD1d^−/− mice compared with WT mice. Based on these findings, our results suggest that NKT cells play a regulatory role that helps to prevent diet-induced obesity and metabolic dysfunction and may play an important role in mechanisms governing cross-talk between metabolism and the immune system to regulate energy balance and liver health.     

非酒精性脂肪性肝病与代谢综合征组分的相关性研究进展

非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)在全世界人群中发病率逐年上升,成为新的全球公共健康问题,已越来越引起临床关注。其发病以胰岛素抵抗为基础,与肥胖、血脂紊乱、原发性高血压、2型糖尿病等代谢综合征各组分密切相关。NAFLD可进展至肝硬化、肝衰竭甚至肝癌,伴有代谢综合征一种或多种组分可能加速疾病的进展。NAFLD初期是一种可逆性过程,充分了解影响其发生、发展的相关代谢危险因素并及时纠正,可能导致疾病的逆转或延缓其进程。本文就NAFLD与代谢综合征各组分的相关调查研究进行综述。     

2 型糖尿病合并非酒精性脂肪肝病患者IGF-1 与胰岛素抵抗关系研究

研究胰岛素样生长因子-1(IGF-1)与2型糖尿病(T2DM)胰岛素抵抗关系。有研究证实给予IGF-1后,可改善胰岛素抵抗、肝脏脂质代谢,IGF-1基因缺失的动物会产生胰岛素抵抗和高胰岛素血症,低水平的IGF-1还可能与非酒精性脂肪肝病(NAFLD)肝纤维化有关,而T2DM和NAFLD与胰岛素抵抗共存,T2DM合并NAFLD患者IGF-1水平更低。IGF-1与胰岛素抵抗关系密切,IGF-1水平能反映胰岛素抵抗的严重程度,为IGF-l在今后治疗T2DM和NAFLD的提供了潜在的临床应用前景。     

Mitochondrial dysfunction in non-alcoholic fatty liver disease and insulin resistance: Cause or consequence?

Abstract

Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of the metabolic syndrome and refers to a spectrum of disorders ranging from steatosis to steatohepatitis, a disease stage characterized by inflammation, fibrosis, cell death and insulin resistance (IR). Due to its association with obesity and IR the impact of NAFLD is growing worldwide. Consistent with the role of mitochondria in fatty acid (FA) metabolism, impaired mitochondrial function is thought to contribute to NAFLD and IR. Indeed, mitochondrial dysfunction and impaired mitochondrial respiratory chain have been described in patients with non-alcoholic steatohepatitis and skeletal muscle of obese patients. However, recent data have provided evidence that pharmacological and genetic models of mitochondrial impairment with reduced electron transport stimulate insulin sensitivity and protect against diet-induced obesity, hepatosteatosis and IR. These beneficial metabolic effects of impaired mitochondrial oxidative phosphorylation may be related not only to the reduction of reactive oxygen species production that regulate insulin signaling but also to decreased mitochondrial FA overload that generate specific metabolites derived from incomplete FA oxidation (FAO) in the TCA cycle. In line with the Randle cycle, reduced mitochondrial FAO rates may alleviate the repression on glucose metabolism in obesity. In addition, the redox paradox in insulin signaling and the delicate mitochondrial antioxidant balance in steatohepatitis add another level of complexity to the role of mitochondria in NAFLD and IR. Thus, better understanding the role of mitochondria in FA metabolism and glucose homeostasis may provide novel strategies for the treatment of NAFLD and IR.     

Biosynthesis and bioavailability of long-chain polyunsaturated fatty acids in non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) has a high occurrence in most countries. Recent studies estimate its prevalence to be near 30% in United States, Italian and Japanese general adult populations. NAFLD commonly presents along with obesity and insulin resistance (IR), pathologies that share with NAFLD metabolic and inflammatory components. These conditions, particularly NAFLD, are associated with alterations in the bioavailability of long-chain polyunsaturated fatty acids (LCPUFAs). In the human population, the bioavailability of LCPUFAs depends both on endogenous biosynthesis and diet amount of preformed LCPUFAs. However, the lower liver LCPUFAs product/precursor ratio namely (20:5n?3 + 22:6n?3)/18:3n?3, 20:4n?6/18:2n?6 present in common Western diets, makes critical an adequate pathway activity to ensure minimum bioavailability of LCPUFAs in most Western populations. The key step of this biosynthesis involves Δ5 and Δ6-desaturases, whose activities are altered in NAFLD. During the disease, the presence of molecular activators of these two enzymes does not correlate with the scarce LCPUFAS biosynthesis observed. The key to this apparent contradiction, or at least part of it, could be explained on the basis of the possible sensitivity of the desaturases to oxidative stress; a metabolic condition strongly linked to inflammatory pathologies such as NAFLD, obesity and IR and that, according to latest research, not only would be consequence but also possibly a cause of these diseases. The present review is focused on the relationship between NAFLD and the bioavailability of LCPUFAs, with special reference to the role that oxidative stress could play in the modulation of the liver fatty acid desaturase activity.     

Loss and gain of function of Grp75 or mitofusin 2 distinctly alter cholesterol metabolism,but all promote triglyceride accumulation in hepatocytes

In the liver, contact sites between the endoplasmic reticulum (ER) and mitochondria (named MAMs) may be crucial hubs for the regulation of lipid metabolism, thus contributing to the exacerbation or prevention of fatty liver. We hypothesized that tether proteins located at MAMs could play a key role in preventing triglyceride accumulation in hepatocytes and nonalcoholic fatty liver disease (NAFLD) occurrence. To test this, we explored the role of two key partners in building MAM integrity and functionality, the glucose-regulated protein 75 (Grp75) and mitofusin 2 (Mfn2), which liver contents are altered in obesity and NAFLD. Grp75 or Mfn2 expression was either silenced using siRNA or overexpressed with adenoviruses in Huh7 cells.Silencing of Grp75 and Mfn2 resulted in decreased ER-mitochondria interactions, mitochondrial network fusion state and mitochondrial oxidative capacity, while overexpression of the two proteins induced mirror impacts on these parameters. Furthermore, Grp75 or Mfn2 silencing decreased cellular cholesterol content and enhanced triglyceride secretion in ApoB100 lipoproteins, while their overexpression led to reverse effects. Cellular phosphatidylcholine/phosphatidylethanolamine ratio was decreased only upon overexpression of the proteins, potentially contributing to altered ApoB100 assembly and secretion. Despite the opposite differences, both silencing and overexpression of Grp75 or Mfn2 induced triglyceride storage, although a fatty acid challenge was required to express the alteration upon protein silencing. Among the mechanisms potentially involved in this phenotype, ER stress was closely associated with altered triglyceride metabolism after Grp75 or Mfn2 overexpression, while blunted mitochondrial FA oxidation capacity may be the main defect causing triglyceride accumulation upon Grp75 or Mfn2 silencing. Further studies are required to decipher the link between modulation of Grp75 or Mfn2 expression, change in MAM integrity and alteration of cholesterol content of the cell.In conclusion, Grp75 or Mfn2 silencing and overexpression in Huh7 cells contribute to altering MAM integrity and cholesterol storage in opposite directions, but all promote triglyceride accumulation through distinct cellular pathways. This study also highlights that besides Mfn2, Grp75 could play a central role in hepatic lipid and cholesterol metabolism in obesity and NAFLD.     

Roles of PPARs in NAFLD: potential therapeutic targets

Non-alcoholic fatty liver disease (NAFLD) is a liver pathology with increasing prevalence due to the obesity epidemic. Hence, NAFLD represents a rising threat to public health. Currently, no effective treatments are available to treat NAFLD and its complications such as cirrhosis and liver cancer. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors which regulate lipid and glucose metabolism as well as inflammation. Here we review recent findings on the pathophysiological role of PPARs in the different stages of NAFLD, from steatosis development to steatohepatitis and fibrosis, as well as the preclinical and clinical evidence for potential therapeutical use of PPAR agonists in the treatment of NAFLD. PPARs play a role in modulating hepatic triglyceride accumulation, a hallmark of the development of NAFLD. Moreover, PPARs may also influence the evolution of reversible steatosis toward irreversible, more advanced lesions. Presently, large controlled trials of long duration are needed to assess the long-term clinical benefits of PPAR agonists in humans. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.