高脂饮食诱导非酒精性脂肪性肝病动物模型的研究进展

非酒精性脂肪性肝病(NAFLD)是以肝细胞脂肪变性为主要特征,但无过量饮酒史的临床病理综合征.研究证实,通过胆固醇和/或脂肪为主的高脂饮食可建立NAFLD的动物模型.然而,在动物选择、高脂饮食配方、喂食条件、造模时间、肝脏病变程度等方面仍需进行深入探索.此文对相关的研究进展作一综述.     

间断注射骨钙素可预防高脂饮食诱导小鼠非酒精性脂肪性肝病的发生

目的：探讨外源性骨钙素干预是否可预防高脂饮食诱导的野生型小鼠非酒精性脂肪性肝病（NAFLD）的发生。方法将5周龄C57BL/6J雄性小鼠随机分为4组：（1）普通饮食对照组;（2）高脂饮食对照组;（3）高脂饮食＋骨钙素低剂量组;（4）高脂饮食＋骨钙素高剂量组。实验期间监测体质量、血糖、进食量等指标;实验结束后,检测血清总胆固醇（TC）、甘油三酯（TG）、游离脂肪酸（FFA）、肿瘤坏死因子α（TNF-α）等指标,并行肝组织学检查及肝TC、TG含量测定。HE染色和油红O染色观察肝形态学改变。结果（1）与高脂饮食对照组相比,两种不同剂量骨钙素干预组的体内脂肪含量,包括附睾旁脂肪、肾周脂肪、皮下脂肪及脂体比均明显降低（均P＜0.001）;空腹血糖及葡萄糖耐量较高脂对照组明显改善（P＜0.05～0.001）;血清TC、TG、FFA、TNF-α等指标较高脂对照组明显降低（均P＜0.05）;（2）两种不同剂量骨钙素干预组的肝脂肪沉积明显减少,肝质量、肝体比、肝TC及TG含量等指标均较高脂饮食对照组明显降低（均P＜0.05）。结论骨钙素间断注射可有效预防高脂饮食诱导小鼠NAFLD的发生,改善糖脂代谢,并可降低血清TNF-α水平。     

Metabolic liver disease of obesity and role of adipose tissue in the pathogenesis of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease （NAFLD） is an increasingly recognized cause of liver-related morbidity and mortality. It can develop secondary to numerous causes but a great majority of NAFLD cases occur in patients who are obese or present with other components of metabolic syndrome （hypertension, dyslipidemia, diabetes）. This is called primary NAFLD and insulin resistance plays a key role in its pathogenesis. Obesity is characterized by expanded adipose tissue, which is under a state of chronic inflammation. This disturbs the normal storage and endocrine functions of adipose tissue. In obesity, the secretome （adipokines, oytokines, free fatty acids and other lipid moieties） of fatty tissue is amplified, which through its autocrine, paracrine actions in fat tissue and systemic effects especially in the liver leads to an altered metabolic state with insulin resistance （IR）. IR leads to hyperglycemia and reactive hyperinsulinemia, which stimulates lipid-accumulating processes and impairs hepatic lipid metabolism. IR enhances free fatty acid delivery to liver from the adipose tissue storage due to uninhibited lipolysis. These changes result in hepatic abnormal fat accumulation, which may initiate the hepatic IR and further aggravate the altered metabolic state of whole body. Hepatic steatosis can also be explained by the fact that there is enhanced dietary fat delivery and physical inactivity. IR and NAFLD are also seen in various lipodystrophic states in contrary to popular belief that these problems only occur due to excessive adiposity in obesity. Hence, altered physiology of adipose tissue is central to development of IR, metabolic syndrome and NAFLD.     

Visceral adipose tissue visfatin in nonalcoholic fatty liver disease

Background. Visfatin is a novel adipocytokine predominantly expressed and secreted by visceral adipose tissue. It is realized for its multiple functions of central importance in NAD biosynthesis, innate immunity and inflammation. Its phosphoribosyl transferase activity regulates cellular energetics and NAD dependent enzymes such as SIRTUINS. Although its expression in various tissues and circulating levels are documented, visceral visfatin levels in Nonalcoholic fatty liver disease (NAFLD) patients have not been reported.Objective. The aim of the present study was to assess visceral adipose tissue visfatin levels in NAFLD.Materials and methods. A total of 115 patients undergoing diagnostic laparoscopy were recruited in the study and categorized into two groups based on standard criteria for NAFLD. Visceral adipose tissue TNF-α, IL-6 and visfatin levels were measured by ELISA. Blood glucose, lipids, liver enzymes and non esterified fatty acids (NEFA) were estimated using standard procedures. Formalin fixed, Hematoxylene Eosin stained liver biopsy specimens were examined for the presence of steatosis and the degree of steatosis was ascertained as per Brunt’s classification.Results. The visceral visfatin level declined significantly (P < 0.001) in all groups of NAFLD as compared to non NAFLD group, while plasma NEFA level increased with progressive steatosis (P < 0.02). Significant increase in TNF a was observed in all groups of NAFLD, while IL-6 increased in NASH only.Conclusion. A significant decline in visceral adipose tissue visfatin level was found to be associated with degree of steatosis in NAFLD patients.     

Melatonin attenuates high fat diet-induced fatty liver disease in rats

AIM:To investigate melatonin’s preventive action in oxidative stress in a rat model with high fat diet-induced non-alcoholic fatty liver disease(NAFLD).METHODS:NAFLD was induced by high fat diet(HFD)in adult,male,Wistar rats,weighing 180-230 g.After acclimatization for one week,they were randomly assigned to 6 experimental groups that comprised animals on regular diet plus 5 or 10 mg/kg melatonin,for 4 or 8 wk;animals on HFD,with or without 5 or 10 mg/kg melatonin,for 4 or 8 wk;and animals on HFD for 8 or 12 wk,with melatonin 10 mg/kg for the last 4 wk.Liver damage was assessed biochemically by the serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and histologically.Lipid peroxidation and oxidative stress were assessed by malondialdehyde and glutathione levels in liver tissue.Lipidemic indices and portal vein pressure were also measured.RESULTS:Compared to rats not receiving melatonin,rats on 5 or 10 mg/kg of melatonin had lower mean liver weight(-5.0 g and-4.9 g)(P < 0.001)and lower liver weight to body weight ratio(-1.0%)(P < 0.001),for the two doses,respectively.All rats fed HFD without melatonin developed severe,grade Ⅲ,steatosis.Rats on HFD with concurrent use of melatonin showed significantly less steatosis,with grade Ⅲ steatosis observed in 1 of 29(3.4%)rats on 10 mg/kg melatonin and in 3 of 27(11.1%)rats on 5 mg/kg melatonin.Melatonin was ineffective in reversing established steatosis.Melatonin also had no effect on any of the common lipidemic serum markers,the levels of which did not differ significantly among the rats on HFD,irrespective of the use or not of melatonin.Liver cell necrosis was significantly less in rats on HFD receiving melatonin than in those not on melatonin,with the AST levels declining by a mean of 170 U/L(P = 0.01)and 224 U/L(P = 0.001),and the ALT levels declining by a mean of 62.9 U/L(P = 0.01)and 93.4 U/L(P < 0.001),for the 5 and 10 mg/kg melatonin dose,respectively.Melatonin mitigated liver damage due to peroxidation and oxidative stress in liver tissue as indicated by a significant decline in MDA production by 12.7(P < 0.001)and 12.2(P < 0.001)μmol/L/mg protein/mg tissue,and a significant increase in glutathione by 20.1(P = 0.004)and 29.2(P < 0.001)μmol/L/mg protein/mg tissue,for the 5 and 10 mg/kg melatonin dose,respectively.CONCLUSION:Melatonin can attenuate oxidative stress,lessen liver damage,and improve liver histology in rats with high fat diet-induced NAFLD,when given concurrently with the diet.     

Deletion of PPARgamma in adipose tissues of mice protects against high fat diet-induced obesity and insulin resistance

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a crucial role in adipocyte differentiation, glucose metabolism, and other physiological processes. To further explore the role of PPARgamma in adipose tissues, we used a Cre/loxP strategy to generate adipose-specific PPARgamma knockout mice. These animals exhibited marked abnormalities in the formation and function of both brown and white adipose tissues. When fed a high-fat diet, adipose-specific PPARgamma knockout mice displayed diminished weight gain despite hyperphagia, had diminished serum concentrations of both leptin and adiponectin, and did not develop glucose intolerance or insulin resistance. Characterization of in vivo glucose dynamics pointed to improved hepatic glucose metabolism as the basis for preventing high-fat diet-induced insulin resistance. Our findings further illustrate the essential role for PPARgamma in the development of adipose tissues and suggest that a compensatory induction of hepatic PPARgamma may stimulate an increase in glucose disposal by the liver.     

Freeze-dried Si-Ni-San powder can ameliorate high fat diet-induced non-alcoholic fatty liver disease

BACKGROUND Non-alcoholic fatty liver disease(NAFLD)is a common chronic liver disease worldwide.However,to date,there is no ideal therapy for this disease.AIM To study the effects of Si-Ni-San freeze-dried powder on high fat diet-induced NAFLD in mice.METHODS Twenty-four male C57BL/6 mice were randomized into three groups of eight.The control group(CON)was allowed ad libitum access to a normal chow diet.The high fat diet group(FAT)and Si-Ni-San group(SNS)were allowed ad libitum access to a high fat diet.The SNS group was intragastrically administered Si-Ni-San freeze-dried powder(5.0 g/kg)once daily,and the CON and FAT groups were intragastrically administered distilled water.After 12 wk,body weight,liver index,visceral fat index,serum alanine aminotransferase(ALT),portal lipopoly-saccharide(LPS),liver tumor necrosis factor(TNF)-αand liver triglycerides were measured.Intestinal microbiota were analyzed using a 16S r DNA sequencing technique.RESULTS Compared with the FAT group,the SNS group exhibited decreased body weight,liver index,visceral fat index,serum ALT,portal LPS,liver TNF-αand liver triglycerides(P<0.05).Intestinal microbiota analysis showed that the SNS group had different bacterial composition and function compared with the FAT group.In particular,Oscillospira genus was a bacterial biomarker of SNS group samples.CONCLUSION The beneficial effects of Si-Ni-San freeze-dried powder on high fat diet-induced NAFLD in mice may be associated with its anti-inflammatory and changing intestinal microbiota effects.     

Role of pentoxifylline in non-alcoholic fatty liver disease in high-fat diet-induced obesity in mice

AIM:To study pentoxifylline effects in liver and adipose tissue inflammation in obese mice induced by high-fat diet(HFD).METHODS: Male swiss mice(6-wk old) were fed a highfat diet(HFD; 60% kcal from fat) or AIN-93(control diet; 15% kcal from fat) for 12 wk and received pentoxifylline intraperitoneally(100 mg/kg per day) for the last 14 d. Glucose homeostasis was evaluated by measurements of basal glucose blood levels and insulin tolerance test two days before the end of the protocol. Final body weight was assessed. Epididymal adipose tissue was collected and weighted for adiposity evaluation. Liver and adipose tissue biopsies were homogenized in solubilization buffer and cytokines were measured in supernatant by enzyme immunoassay or multiplex kit, respectively. Hepatic histopathologic analyses were performed in sections of paraformaldehyde-fixed, paraffin-embedded liver specimens stained with hematoxylin-eosin by an independent pathologist. Steatosis(macrovesicular and microvesicular), ballooning degeneration and inflammation were histopathologically determined. Triglycerides measurements were performed after lipid extraction in liver tissue. RESULTS: Pentoxifylline treatment reduced microsteatosis and tumor necrosis factor(TNF)-α in liver(156.3 ± 17.2 and 62.6 ± 7.6 pg/mL of TNF-α for non-treated and treated obese mice, respectively; P 0.05). Serum aspartate aminotransferase levels were also reduced(23.2 ± 6.9 and 12.1 ± 1.6 U/L for nontreated and treated obese mice, respectively; P 0.05) but had no effect on glucose homeostasis. In obese adipose tissue, pentoxifylline reduced TNF-α(106.1 ± 17.6 and 51.1 ± 9.6 pg/mL for non-treated and treated obese mice, respectively; P 0.05) and interleukin-6(340.8 ± 51.3 and 166.6 ± 22.5 pg/mL for non-treated and treated obese mice, respectively; P 0.05) levels; however, leptin(8.1 ± 0.7 and 23.1 ± 2.9 ng/mL for non-treated and treated lean mice, respectively; P 0.05) and plasminogen activator inhibitor-1(600.2 ± 32.3 and 1508.6 ± 210.4 pg/mL for non-treated and treated lean mice, respectively; P 0.05) levels increased in lean adipose tissue. TNF-α level in the liver of lean mice also increased(29.6 ± 6.6 and 75.4 ± 12.6 pg/mL for non-treated and treated lean mice, respectively; P 0.05) while triglycerides presented a tendency to reduction.CONCLUSION: Pentoxifylline was beneficial in obese mice improving liver and adipose tissue inflammation. Unexpectedly, pentoxifylline increased pro-inflammatory markers in the liver and adipose tissue of lean mice.     

Correlation of adipose tissue with liver histology in Asian Indian patients with nonalcoholic fatty liver disease (NAFLD)

Background. There is sparse literature on the association of adipose tissue with liver histology in patients with nonalcoholic fatty liver disease (NAFLD).Aim. To study the correlation of visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT) and total adipose tissue (TAT) with liver histology in Indian patients with NAFLD.Material and methods. A single slice CT scan at the level of L4-L5 vertebrae was done to assess the abdominal VAT and SAT volumes in 21 patients with histological diagnosis of NAFLD. Adult treatment panel III criteria with modified abnormal waist were used to define metabolic syndrome (MS). Histological grading was done according to the NAFLD activity score (NAS).Results. Twenty-one patients with NAFLD [13 males, median age: 35 years, median BMI: 25.97 kg/m²] were included prospectively. Even though overweight/obese patients had severe liver disease, there was no difference in the volume of VAT adjusted for BMI between 6 (28.5%) lean and 15 (71.5%) overweight/obese patients. Patients with NASH and borderline NASH were older, obese with higher VAT and SAT volumes than no-NASH group. SAT volume (SATV) correlated significantly with hepatic steatosis but none of the adipose tissue volumes had any correlation with other histological variables. Both SATV and TAT volume (TATV) correlated significantly with severity of liver disease as determined by NAS score whereas presence of MS or insulin resistance had no correlation with histological severity.Conclusion. Both subcutaneous and total adipose tissue volume are related to the disease severity as determined by NAFLD activity score in Indian patients with NAFLD.     

肝脏脂肪酸在高脂饮食大鼠非酒精性脂肪性肝病模型中的代谢

目的: 探索脂肪酸代谢变化在非酒精性脂肪性肝病(non-alcoholic fatty liver disease, NAFLD)发病中的作用.方法:SD大鼠正常喂养1 wk后,随机分为正常组和高脂饮食组(n=7).正常组大鼠以普通饲料喂养,高脂饮食组以高脂饲料喂养.实验第16周分别处死大鼠,观察肝组织的病理改变.ECF衍生GC/MS技术分析NASH大鼠与正常大鼠肝组织脂肪酸代谢的变化,并运用SPSS11.0软件,进行主成分分析(PCA),观察代谢产物信息.结果:高脂饮食组大鼠脂肪肝病变程度均为 ,均达到脂肪肝诊断标准,且炎症明显.NASH和正常组大鼠肝组织代谢组形态具有明显差异.高脂饮食大鼠NASH肝脏与正常组相比较,存在明显的脂肪酸谱变化.与对照组相比.除正十二烷酸降低0.56,其余脂肪酸均显著升高(P<0.05),硬脂酸、十四烷酸、十六烷酸、二十烷酸、油酸、α-亚麻酸、花生四烯酸和亚油酸分别升高5.42、4.10、11.56、5.86、1.89、1.82、8.00和2.44倍.且ω-6/ω-3多不饱和脂肪酸的比值升高.结论:NASH大鼠肝脏存在明显的脂肪酸代谢变化,肝细胞内各类脂肪酸聚积、ω-6/ω-3多不饱和脂肪酸比率失衡和正十二烷酸降低可能对脂肪肝炎症发生、肝损伤有重要作用.     

Visceral fat accumulation and insulin resistance are important factors in nonalcoholic fatty liver disease

Background Nonalcoholic fatty liver diseases are often associated with obesity, insulin resistance, and excessive visceral fat accumulation. The aims of this study were (1) to evaluate the relationship between the severity of fatty liver and visceral fat accumulation in nonalcoholic fatty liver diseases, and (2) to investigate the relationships of fatty liver with biochemical data and insulin resistance. Methods One hundred twenty-nine subjects (63 women) with fatty liver diagnosed by ultrasonography were enrolled. Subjects positive for hepatitis B virus, hepatitis C virus, or autoimmune antibodies and those whose alcohol intake was over 20 g/day were excluded. The visceral fat area at the umbilical level and the liver–spleen ratio were evaluated by computed tomography. Results The severity of fatty liver evaluated by ultrasonography showed a significant positive relationship with the visceral fat area and waist circumstance (fatty liver severity: mild, 92.0 ± 30.9 cm²; moderate, 122.1 ± 32.6 cm²; severe, 161.0 ± 48.4 cm²; P < 0.0001). The visceral fat area and liver–spleen ratio were negatively correlated (r = −0.605, P < 0.0001). The severity of fatty liver showed strong positive relationships with serum aspartate aminotransferase, alanine aminotransferase, fasting plasma glucose, fasting plasma insulin, and insulin resistance. The severity of fatty liver was positively related to the visceral fat area in 49 nonobese subjects (body mass index <25). Conclusions The severity of fatty liver was positively correlated with visceral fat accumulation and insulin resistance in both obese and nonobese subjects, suggesting that hepatic fat infiltration in nonalcoholic fatty liver disease may be influenced by visceral fat accumulation regardless of body mass index.     

吡格列酮对肥胖小鼠原代棕色脂肪细胞分化和功能基因表达的影响

观察吡格列酮对肥胖小鼠原代棕色脂肪(brown adipose tissue,BAT)细胞分化和功能基因表达的影响,并为治疗肥胖相关疾病寻找新思路.结果显示吡格列酮通过增强特异基因表达、促进分化成脂、提高线粒体功能等方面增强BAT细胞功能(P＜0.05);这可能是吡格列酮改善机体代谢的重要原因.     

The obesity epidemic and nonalcoholic fatty liver disease in children

Childhood obesity is a worldwide health problem associated with an increase in the prevalence and severity of nonalcoholic fatty liver disease (NAFLD). This review covers the progress made between 2005 and 2007 in understanding the epidemiology, histology, and treatment of pediatric NAFLD. The number of children with NAFLD presents a major public health crisis. Noninvasive diagnostic tools offer future promise, but currently are unable to grade and stage disease. Therefore, pediatric NAFLD remains a clinico-pathological diagnosis requiring direct demonstration of liver steatosis and the exclusion of other causes of fatty liver and/or hepatitis. There are currently no proven therapies for NAFLD in children; however, TONIC (Treatment of Nonalcoholic Fatty Liver Disease in Children), the first multicenter clinical trial of pediatric NAFLD, is currently in progress. Such studies are imperative to address fundamental questions regarding cause and cure.     

Visceral adipose tissue quality was associated with nonalcoholic fatty liver disease,independent of its quantity

Background and aimsNonalcoholic fatty liver disease (NAFLD) is a serious liver disease. Recent studies have shown that both visceral adipose tissue (VAT) quantity and density (as an indirect measure of quality) are associated with metabolic profiles. Therefore, we investigated the association between VAT quantity and quality, and the prevalence and incidence of NAFLD.Methods and resultsIn this cross-sectional, retrospective cohort study, the prevalence and incidence of NAFLD were analyzed in 627 and 360 middle-aged subjects, respectively. VAT was evaluated using an unenhanced computed tomography scan, while NAFLD was evaluated using ultrasonography. The VAT area was normalized to the square value of the subjects’ height in meters, the visceral fat area (VFA) index. The VAT density was described as the visceral fat density (VFD). The VFA index and VFD had an interaction effect on the prevalence of NAFLD (P = 0.0059). The VFA index (adjusted odds ratio [OR], 1.04; 95% confidence interval [CI], 1.01–1.07; P = 0.0145, per 1.0 cm²/m²) and the VFD (OR, 0.90; 95% CI, 0.84–0.96; P = 0.0026, per 1.0 Hounsfield unit [HU]) were independently associated with the prevalence of NAFLD. In our cohort, 36 subjects developed NAFLD. The VFD (adjusted hazards ratio [HR], 0.84; 95% CI, 0.77–0.91; P < 0.0001, per 1.0 HU) was independently associated with the incidence of NAFLD, whereas the VFA index was not.ConclusionBoth the VFA index and VFD were independently associated with NAFLD prevalence. The VFD might be more related to the incidence of NAFLD than the VFA index.