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Betulin prevents high fat diet-induced non-alcoholic fatty liver disease by mitigating oxidative stress and upregulating Nrf2 and SIRT1 in rats
Abstract
Non-alcoholic fatty liver disease (NAFLD) is a common chronic hepatic disorder characterized by hepatic lipid accumulation. This study explored the effect of betulin (BE), a terpenoid with promising antioxidant, anti-inflammatory and insulin sensitizing effects, on NAFLD induced by high fat diet (HFD). Rats received HFD and BE (15 and 30 mg/kg) for 12 weeks and blood and liver samples were collected for analyses. HFD caused hyperlipidemia, cholesterol and triglycerides accumulation in the liver, hepatocellular ballooning, fibrosis, insulin resistance (IR), lipid peroxidation (LPO), and NF-kB p65 upregulation. BE ameliorated serum and liver lipids, blood glucose, and insulin, liver LPO, prevented steatosis and fibrosis, suppressed NF-kB p65 and enhanced antioxidants in HFD-fed rats. BE downregulated ACC1 and FAS, and upregulated Nrf2, HO-1 and SIRT1 in the liver of HFD-fed rats. In silico investigations revealed the binding affinity of BE towards NF-kB, Keap1, HO-1 and SIRT1. In conclusion, BE attenuated HFD-induced NAFLD by ameliorating hyperlipidemia, IR, lipogenesis, liver lipid accumulation, and oxidative stress. The protective effect of BE was associated with enhanced Nrf2/HO-1 signaling and SIRT1.
... In this context, Nazir et al. [43] documented that Daphne mucronata Royle exhibit antioxidant properties in the paracetamol-induced hepatotoxicity by reducing lipid peroxidation, which was characterized by a decrease in the MDA level. Furthermore, Betulin is reported to be a direct scavenger of free oxygen radicals in high-fat diet-induced non-alcoholic fatty liver disease by mitigating oxidative stress [44]. Buko et al. also showed that Betulin effectively improves alcohol-induced liver damage by reducing superoxide anion in liver mitochondria and reducing MDA levels in the liver tissue [45]. ...
Background and objective
Pulmonary fibrosis (PF) is a chronic and progressive respiratory disease representing the final stage of lung inflammatory disorders. Reactive oxygen species (ROS), an essential factor in the formation and progression of pulmonary fibrosis, are a significant adverse effect of Bleomycin (BLM). Antioxidant activities have been found in Daphne oleoides. In this study, we attempted to explore the function of hydroalcoholic extract of Daphne oleoides (D. oleoides) and Betulin in inhibiting bleomycin (BLM)-induced pulmonary fibrosis in rat".
Materials and methods
The current experimental study used 36 male Wistar rats (180–220). Following a random process, the animals were divided into six groups six (n = 6). Group, I (the control group) received normal saline, while Group II (the hazardous group) received intratracheal BLM (7.5 units per kg). Following the administration of BLM, Groups V and VI received daily doses of vitamin E (500 mg/kg/d, p.o.) and Betulin (10 mg kg/d, p.o.), whereas Groups III and IV received daily doses of Daphne oleoides extract (300 and 600 mg/kg/d, p.o.). Then, blood samples from the hearts of the animals were taken to assess the plasma concentrations of nitric oxide (NO) and malondialdehyde (MDA). Finally, the rats were euthanized, and the lung tissues were taken out for histological analysis and assessments of the levels of lung hydroxyproline (HP), ferric-reducing ability (FRAP), NO, Glutathione Concentration (GSH), thiol content (tSH) and MDA.
Findings
Elevated lung index, lung hydroxyproline, NO, and MDA plasma levels, and a reduction in total body thiol content (tSH) in the group receiving BLM were evidence of pulmonary toxicity. Treatment with D. oleoides extracts, Betulin, and Vit E, especially at 600 mg/kg, led to a marked reduction in the above parameters compared with the BLM-received group (p < 0.01). Histological Analysis of the BLM-treated group showed a considerable Lung injury with interstitial infiltration, collapsed alveolar spaces, and alveolar septal thickening. These changes were mitigated with D. oleoides 600, Betulin-, and vitamin E. These changes were mitigated with D. oleoides 600, Betulin-, and vitamin E.
Conclusion
These findings suggest that D. oleoides and Betulin prevent bleomycin-induced lung fibrosis in rats by decreasing inflammatory and antioxidant markers. Daphne oleoides, therefore, have the potential to be used therapeutically to treat pulmonary fibrosis.
Betulin is a heavily studied natural compound for its use as an anticancer or pro-regenerative agent. The structural similarity between betulin to steroids gives rise to the idea that the substance may as well act as an anti-inflammatory drug. This study is the first to describe the anti-inflammatory properties of betulinic acid, betulin, and its derivatives with amino acids 1,4-diaminebutane (Dab), 1,3-diaminepropane (Dap), Ornithine (Orn), and lysine (Lys) on murine macrophages from lymphoma site. The compounds were compared to dexamethasone. To establish the response of the macrophages to the natural compounds, we tested the viability as well as sensitivity to the inflammatory signaling (IFNγR). IL-6 secretory properties and HSP-70 content in the cells were examined. Furthermore , we characterized the effects of compounds on the inhibition of cyclooxygenase-2 (COX-2) activity both in the enzymatic assays and molecular docking studies. Then, the changes in COX-2 expression after betulin treatment were assessed. Betulin and betulinic acid are the low-cytotoxicity compounds with the highest potential to decrease inflammation via reduced IL-6 secretion. To some extent, they induce the reorganization of IFNγR with nearly no effect on COX-2 activity. Conversely, Bet-Orn and Bet-Lys are highly Szlasa et al. cytotoxic and induce the aggregation of IFNγR. Besides, Bet-Lys reduces the activity of COX-2 to a higher degree than dexamethasone. Bet-Orn is the only one to increase the HSP-70 content in the macrophages. In case of IL-6 reduction, all compounds were more potent than dexamethasone.
The purpose of this study was to investigate the protective effect of sniffing orange essential oil (OEO) on the formation of non-alcoholic fatty liver disease (NAFLD) caused by a high-fat diet. The results confirmed that sniffing OEO could reduce obesity caused by a high-fat diet (HFD) by reducing the levels of triglycerides (TGs), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). In addition, the observation of liver tissue sections showed that sniffing OEO could reduce lipid accumulation in liver cells. Further analysis by western blot analysis showed that OEO treatment made the expression levels of acetyl-CoA carboxylase (ACC) and Cytochrome P450 2E1 (CYP2E1) down-regulated and the expression levels of peroxisome proliferator-activated receptor-α (PPAR-α) and carnitine palmitoyltransferase-1 (CPT-1) up-regulated. These results indicate that the treatment of sniffing OEO could enhance the antioxidant capacity of mice and reduce liver damage caused by a high-fat diet. Furthermore, sniffing OEO could inhibit lipid synthesis and oxidative stress stimulated by a high-fat diet. Overall, OEO treatment had a certain protective effect on NAFLD-related diseases caused by a high-fat diet. Therefore, aromatherapy may be introduced as a treatment of long-term chronic diseases.
Non-alcoholic fatty liver disease (NAFLD) is a challenging disease caused by multiple factors, which may partly explain why it remains still orphan of an adequate therapeutic strategy. Herein we focus on the interplay between oxidative stress (OS) and the other causal pathogenetic factors. Different reactive oxygen species (ROS) generators contribute to NAFLD inflammatory and fibrotic progression, which is quite strictly linked to the lipotoxic liver injury from fatty acids and/or a wide variety of their biologically active metabolites in the context of either a two-hit or a (more recent) multiple parallel hits theory. An antioxidant defense system is usually able to protect hepatic cells from damaging effects caused by ROS, including those produced into the gastrointestinal tract, i.e., by-products generated by usual cellular metabolic processes, normal or dysbiotic microbiota, and/or diet through an enhanced gut–liver axis. Oxidative stress originating from the imbalance between ROS generation and antioxidant defenses is under the influence of individual genetic and epigenetic factors as well. Healthy diet and physical activity have been shown to be effective on NAFLD also with antioxidant mechanisms, but compliance to these lifestyles is very low. Among several considered antioxidants, vitamin E has been particularly studied; however, data are still contradictory. Some studies with natural polyphenols proposed for NAFLD prevention and treatment are encouraging. Probiotics, prebiotics, diet, or fecal microbiota transplantation represent new therapeutic approaches targeting the gut microbiota dysbiosis. In the near future, precision medicine taking into consideration genetic or environmental epigenetic risk factors will likely assist in further selecting the treatment that could work best for a specific patient.
A high fat Western-style diet leads to hepatic steatosis that can progress to steatohepatitis and ultimately cirrhosis or liver cancer. The mechanism that leads to the development of steatosis upon nutritional overload is complex and only partially understood. Using click chemistry-based metabolic tracing and microscopy, we study the interaction between Kupffer cells and hepatocytes ex vivo. In the early phase of steatosis, hepatocytes alone do not display significant deviations in fatty acid metabolism. However, in co-cultures or supernatant transfer experiments, we show that tumor necrosis factor (TNF) secretion by Kupffer cells is necessary and sufficient to induce steatosis in hepatocytes, independent of the challenge of hepatocytes with elevated fatty acid levels. We further show that free fatty acid (FFA) or lipopolysaccharide are both able to trigger release of TNF from Kupffer cells. We conclude that Kupffer cells act as the primary sensor for both FFA overload and bacterial lipopolysaccharide, integrate these signals and transmit the information to the hepatocyte via TNF secretion. Hepatocytes react by alteration in lipid metabolism prominently leading to the accumulation of triacylglycerols (TAGs) in lipid droplets, a hallmark of steatosis.
Dyslipidemia is a risk factor for cardiovascular disease, steatohepatitis, and progression of liver disorders. This study investigated the protective effect of farnesol (FAR), a sesquiterpene alcohol, against liver injury in high cholesterol diet (HCD)-fed rats, and its modulatory effect on fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). HCD was supplemented for 10 weeks, and the rats were concurrently treated with FAR. Rats that received HCD exhibited significant elevation of serum cholesterol, triacylglycerols, LDL and vLDL cholesterol, CRP, and pro-inflammatory cytokines and increased values of the cardiovascular risk indices. Serum transaminases, ALP, LDH and CK-MB, and hepatic lipid peroxidation (LPO), cholesterol, and triacylglycerols were increased in HCD-fed rats. Treatment with FAR greatly ameliorated dyslipidemia and liver function, reduced inflammatory mediators, LPO, and hepatic lipid infiltration and enhanced anti-oxidant defenses. FAR suppressed hepatic FAS, ACC, and SREPB-1c mRNA abundance and FAS activity in HDC-fed rats. In addition, molecular docking simulations pinpointed the binding modes of FAR to the active pocket residues of FAS and ACC. In conclusion, FAR possesses a strong anti-hyperlipidemic/anti-hypercholesterolemic activity mediated through its ability to modulate hepatic FAS, ACC, and SREPB-1c. FAR prevented oxidative stress, inflammation, and liver injury induced by HCD. Thus, FAR may represent a promising lipid-lowering agent that can protect against dyslipidemia and its linked metabolic deregulations.
Targeting the gut-liver axis by modulating the gut-microbiome can be a promising therapeutic approach in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate the effects of single species and a combination of Lactobacillus and Pediococcus in NAFLD mice model. Six-week male C57BL/6J mice were divided into 9 groups (n = 10/group; normal, Western diet, and 7 Western diet-strains [10⁹ CFU/g, 8 weeks]). The strains used were L. bulgaricus, L. casei, L. helveticus, P. pentosaceus KID7, and three combinations (1: L. casei+L. helveticus, 2: L. casei+L. helveticus+P. pentosaceus KID7, and 3: L. casei+L. helveticus+L. bulgaricus). Liver/Body weight ratio, serum and stool analysis, liver pathology, and metagenomics by 16S rRNA-sequencing were examined. In the liver/body ratio, L. bulgaricus (5.1 ± 0.5), L. helveticus (5.2 ± 0.4), P. pentosaceus KID7 (5.5 ± 0.5), and combination1 and 2 (4.2 ± 0.6 and 4.8 ± 0.7) showed significant reductions compared with Western (6.2 ± 0.6)(p < 0.001). In terms of cholesterol and steatosis/inflammation/NAFLD activity, all groups except for L. casei were associated with an improvement (p < .05). The elevated level of tumor necrosis factor-α/interleukin-1β (pg/ml) in Western (65.8 ± 7.9/163.8 ± 12.2) was found to be significantly reduced in L. bulgaricus (24.2 ± 1.0/58.9 ± 15.3), L. casei (35.6 ± 2.1/62.9 ± 6.0), L. helveticus (43.4 ± 3.2/53.6 ± 7.5), and P. pentosaceus KID7 (22.9 ± 3.4/59.7 ± 12.2)(p < 0.01). Cytokines were improved in the combination groups. In metagenomics, each strains revealed a different composition and elevated Firmicutes/Bacteroidetes ratio in the western (47.1) was decreased in L. bulgaricus (14.5), L. helveticus (3.0), and P. pentosaceus KID7 (13.3). L. bulgaricus, L. casei, L. helveticus, and P. pentosaceus KID7 supplementation can improve NAFLD-progression by modulating gut-microbiome and inflammatory pathway.
Seaweeds are rich in structurally diverse bioactive compounds with promising therapeutic effects. This study aimed to isolate and identify terpenes from the brown alga Padina pavonia and to investigate its antidiabetic activity, pointing to the possible involvement of peroxisome proliferator-activated receptor (PPAR)γ. Type 2 diabetes was induced by feeding rats a high fat diet (HFD) for 4 weeks followed by injection of 35 mg/kg streptozotocin (STZ). The diabetic rats received P. pavonia extract (PPE; 50, 100 and 200 mg/kg) for 4 weeks and samples were collected for analyses. HFD/STZ-induced rats showed hyperglycemia, dyslipidemia, impaired glucose tolerance, decreased insulin, and increased HbA1c and HOMA-IR. PPE ameliorated hyperglycemia and dyslipidemia, and improved glucose tolerance and insulin sensitivity in diabetic rats. Treatment with PPE increased hepatic hexokinase activity and glycogen, suppressed glucose-6-phosphatase, fructose-1,6-biphosphatase, and glycogen phosphorylase, and attenuated oxidative stress, inflammation, and liver injury and lipid infiltration in HFD/STZ-induced rats. In addition, PPE boosted antioxidants and upregulated PPARγ gene and protein expression in the liver of diabetic rats. Phytochemical investigation resulted in the isolation of six terpenes from PPE and in silico analysis revealed their binding affinity toward PPARγ. In conclusion, P. pavonia-derived terpenes attenuated hyperglycemia, dyslipidemia, oxidative stress, and inflammation, and improved insulin sensitivity and carbohydrate metabolism in type 2 diabetic rats. These beneficial effects are mediated via PPARγ activation. However, further studies to explore the exact mechanisms underlying the antidiabetic effect of PPE are recommended.
Fatty liver diseases, which are commonly associated with high-fat/calorie diet, heavy alcohol consumption and/or other metabolic disorder causes, lead to serious medical concerns worldwide in recent years. It has been demonstrated that metabolic homeostasis disruption is most likely to be responsible for this global epidemic. Sirtuins are a group of conserved nicotinamide adenine dinucleotide (NAD⁺) dependent histone and/or protein deacetylases belonging to the silent information regulator 2 (Sir2) family. Among seven mammalian sirtuins, sirtuin 1 (SIRT 1) is the most extensively studied one and is involved in both alcoholic and nonalcoholic fatty liver diseases. SIRT1 plays beneficial roles in regulating hepatic lipid metabolism, controlling hepatic oxidative stress and mediating hepatic inflammation through deacetylating some transcriptional regulators against the progression of fatty liver diseases. Here we summarize the latest advances of the biological roles of SIRT1 in regulating lipid metabolism, oxidative stress and inflammation in the liver, and discuss the potential of SIRT1 as a therapeutic target for treating alcoholic and nonalcoholic fatty liver diseases.
Free radicals, reactive oxygen/nitrogen species (ROS/RNS), hydrogen sulphide, and hydrogen peroxide play an important role in both intracellular and intercellular signaling; however, their production and quenching need to be closely regulated to prevent cellular damage. An imbalance, due to exogenous sources of free radicals and chronic upregulation of endogenous production, contributes to many pathological conditions including cardiovascular disease and also more general processes involved in aging. Nuclear factor erythroid 2-like 2 (NFE2L2; commonly known as Nrf2) is a transcription factor that plays a major role in the dynamic regulation of a network of antioxidant and cytoprotective genes, through binding to and activating expression of promoters containing the antioxidant response element (ARE). Nrf2 activity is regulated by many mechanisms, suggesting that tight control is necessary for normal cell function and both hypoactivation and hyperactivation of Nrf2 are indicated in playing a role in different aspects of cardiovascular disease. Targeted activation of Nrf2 or downstream genes may prove to be a useful avenue in developing therapeutics to reduce the impact of cardiovascular disease. We will review the current status of Nrf2 and related signaling in cardiovascular disease and its relevance to current and potential treatment strategies.
An observational study describing the number and type of chronic conditions and medications taken by diabetic patients with NAFLD and identifying characteristics that may impact liver disease severity or clinical management.
Adults with type 2 diabetes have a high prevalence of nonalcoholic fatty liver disease (NAFLD) and increased risk of developing advanced liver disease. Appropriate management should consider the characteristics of the diabetic NAFLD population, as comorbid conditions and medications may increase the complexity of treatment strategies.
Diabetic patients with NAFLD at risk of clinically significant liver disease (as assessed by the FIB-4 or NAFLD fibrosis scores) were recruited consecutively from the Endocrine clinic or primary care. Medical conditions, medication history, anthropometric measurements, and laboratory tests were obtained during assessment. NAFLD severity was classified by transient elastography and liver ultrasound into “no advanced disease” (LSM < 8.2 kPa) or “clinically significant liver disease” (LSM ≥ 8.2 kPa).
The most common coexistent chronic conditions were metabolic syndrome (94%), self-reported “depression” (44%), ischaemic heart disease (32%), and obstructive sleep apnoea (32%). Polypharmacy or hyperpolypharmacy was present in 59% and 31% of patients respectively. Elevated LSM (≥ 8.2 kPa) suggesting significant liver disease was present in 37% of this at-risk cohort. Increasing obesity and abdominal girth were both independently associated with likelihood of having significant liver disease.
There is a high burden of multimorbidity and polypharmacy in diabetic NAFLD patients, highlighting the importance of multidisciplinary management to address their complex health care needs and ensure optimal medical treatment.
Non-alcoholic fatty liver disease (NAFLD) is a common clinicopathological condition, encompassing a range
of conditions caused by lipid deposition within liver cells. To date, no approved drugs are available for the treatment of NAFLD, despite the fact that it represents a serious and growing clinical problem in the Western world. Identification of the molecular mechanisms leading to NAFLD-related fat accumulation, mitochondrial dysfunction and oxidative balance impairment facilitates the development of specific interventions aimed at preventing the progression of hepatic steatosis. In this review, we focus our attention on the role of dysfunctions in mitochondrial bioenergetics in the pathogenesis of fatty liver. Major data from the literature about the mitochondrial targeting of some antioxidant molecules as a potential treatment for hepatic steatosis are described and critically analysed. There is ample evidence of the positive effects of several classes of antioxidants, such as polyphenols (i.e., resveratrol, quercetin, coumestrol, anthocyanins, epigallocatechin gallate and curcumin), carotenoids (i.e., lycopene, astaxanthin and fucoxanthin) and glucosinolates (i.e., glucoraphanin, sulforaphane, sinigrin and allyl-isothiocyanate), on the reversion of fatty liver. Although the mechanism of action is not yet fully elucidated, in some cases an indirect interaction with mitochondrial metabolism is expected. We believe that such knowledge will eventually translate into the development of novel therapeutic approaches for fatty liver.
Scope:
Non-alcoholic fatty liver disease (NAFLD) is a condition characterized by an increment in the liver fat content, with a concomitant reduction in the content of n-3-long chain polyunsaturated fatty acids (n-3 LCPUFAs), downregulation of PPAR-α activity, and upregulation of NF-κB activity, effects that induce pro-lipogenic and pro-inflammatory responses. Hydroxytyrosol (HT), a polyphenol with cytoprotective effects present in extra virgin olive oil, improves the cellular antioxidant capacity for activation of transcription factor Nrf2. The objective of this work is to evaluate the molecular adaptations involved in the anti-lipogenic, anti-inflammatory, and anti-oxidant effects of HT supplementation in high-fat diet (HFD)-fed mice.
Methods and results:
Male C57BL/6J mice received (i) control diet (10% fat); (ii) control diet + HT (daily doses of 5 mg per kg body weight), (iii) HFD (60% fat); or (iv) HFD + HT for 12 weeks. HFD-fed mice exhibited (i) liver steatosis; (ii) inflammation; (iii) oxidative stress; and (iv) depletion of n-3 LCPUFAs, together with down-regulation of PPAR-α and Nrf2, and up-regulation of NF-κB. HT supplementation attenuated the metabolic alterations produced by HFD, normalizing the activity of Nrf2, reducing the drop in activity of PPAR-α, and attenuating increment of NF-κB activation.
Conclusion:
Supplementation with HT activating transcription factors PPAR-α and Nrf2, along with the deactivation of NF-κB, may reduce the liver alterations induced in HFD-fed mice.
SIRT1, a homolog of yeast Sir2, is a type III NAD+ dependent histone and protein deacetylase. Previous studies of mice carrying liver specific deletion of exon 4 of the Sirt1 gene revealed opposite responses of mutant mice to a high-fat diet in terms of fatty liver formation, which obscures the function of SRIT1 in liver development and lipid metabolism. To investigate this, we deleted exons 5 and 6 of Sirt1 in the liver by using a Cre-loxP approach. Western blot using an antibody to N-terminal SIRT1 does not detect a truncated protein in the liver of the mutant mice (Sirt1flox5-6/flox5-6;Alb-Cre), suggesting a null mutation for SIRT1 is generated in the liver. Unlike the previously reported phenotypes, the Sirt1flox5-6/flox5-6;Alb-Cre mice develop fatty liver under a normal feeding condition. The disease starts at two months of age and incidence increases as the animals become older, affecting 78% of them when they are over one year of age. We showed that the steatosis is accompanied by altered expression of a number of genes, including increased expression of ChREBP, which acts as one of the central determinants of lipid synthesis in the liver. This data uncovers an important role of SIRT1 in regulating lipid metabolism in the liver, and the SIRT1 mutant mice may serve as an animal model for studying human fatty liver disease and facilitate the development of effective therapeutic approach for the disease.
The SIRT1 deacetylase inhibits fat synthesis and stimulates fat oxidation in response to fasting, but the underlying mechanisms remain unclear. Here we report that SREBP-1c, a key lipogenic activator, is an in vivo target of SIRT1. SIRT1 interaction with SREBP-1c was increased during fasting and decreased upon feeding, and consistently, SREBP-1c acetylation levels were decreased during fasting in mouse liver. Acetylated SREBP-1c levels were also increased in HepG2 cells treated with insulin and glucose to mimic feeding conditions, and down-regulation of p300 by siRNA decreased the acetylation. Depletion of hepatic SIRT1 by adenoviral siRNA increased acetylation of SREBP-1c with increased lipogenic gene expression. Tandem mass spectrometry and mutagenesis studies revealed that SREBP-1c is acetylated by p300 at Lys-289 and Lys-309. Mechanistic studies using acetylation-defective mutants showed that SIRT1 deacetylates and inhibits SREBP-1c transactivation by decreasing its stability and its occupancy at the lipogenic genes. Remarkably, SREBP-1c acetylation levels were elevated in diet-induced obese mice, and hepatic overexpression of SIRT1 or treatment with resveratrol, a SIRT1 activator, daily for 1 week decreased acetylated SREBP-1c levels with beneficial functional outcomes. These results demonstrate an intriguing connection between elevated SREBP-1c acetylation and increased lipogenic gene expression, suggesting that abnormally elevated SREBP-1c acetylation increases SREBP-1c lipogenic activity in obese mice. Reducing acetylation of SREBP-1c by targeting SIRT1 may be useful for treating metabolic disorders, including fatty liver, obesity, and type II diabetes.
The transcription factor Nrf2 regulates expression of multiple cellular defence proteins through the antioxidant response element (ARE). Nrf2-deficient mice (Nrf2(-/-)) are highly susceptible to xenobiotic-mediated toxicity, but the precise molecular basis of enhanced toxicity is unknown. Oligonucleotide array studies suggest that a wide range of gene products is altered constitutively, however no equivalent proteomics analyses have been conducted. To define the range of Nrf2-regulated proteins at the constitutive level, protein expression profiling of livers from Nrf2(-/-) and wild type mice was conducted using both stable isotope labelling (iTRAQ) and gel electrophoresis methods. To establish a robust reproducible list of Nrf2-dependent proteins, three independent groups of mice were analysed. Correlative network analysis (MetaCore) identified two predominant groups of Nrf2-regulated proteins. As expected, one group comprised proteins involved in phase II drug metabolism, which were down-regulated in the absence of Nrf2. Surprisingly, the most profound changes were observed amongst proteins involved in the synthesis and metabolism of fatty acids and other lipids. Importantly, we show here for the first time, that the enzyme ATP-citrate lyase, responsible for acetyl-CoA production, is negatively regulated by Nrf2. This latter finding suggests that Nrf2 is a major regulator of cellular lipid disposition in the liver.
Mammalian sirtuin 1 (SIRT1) may control fatty acid homeostasis in liver. However, this possibility and underlying mechanism remain to be established. In this study, we addressed the issues by examining the metabolic phenotypes of SIRT1 heterozygous knockout (SIRT1(+/-)) mice. The study was conducted in the mice on three different diets including a low-fat diet (5% fat wt/wt), mediate-fat diet (11% fat wt/wt), and high-fat diet (HFD, 36% fat wt/wt). On low-fat diet, the mice did not exhibit any abnormality. On mediate-fat diet, the mice exhibited a significant increase in hepatic steatosis with elevated liver/body ratio, liver size, liver lipid (triglyceride, glycerol, and cholesterol) content, and liver inflammation. The hepatic steatosis was deteriorated in the mice by HFD. In the liver, lipogenesis was increased, fat export was reduced, and beta-oxidation was not significantly changed. Body weight and fat content were increased in response to the dietary fat. Fat was mainly increased in sc adipose tissue and liver. Inflammation was also elevated in epididymal fat. Whole body energy expenditure and substrate utilization were reduced. Food intake, locomotor activity, and fat absorption were not changed. These data suggest that a reduction in the SIRT1 activity increases the risk of fatty liver in response to dietary fat. The liver steatosis may be a result of increased lipogenesis and reduced liver fat export. The inflammation may contribute to the pathogenesis of hepatic steatosis as well. A reduction in lipid mobilization may contribute to the hepatic steatosis and low energy expenditure.
Diabetic nephropathy (DN) is a serious complication of diabetes and can lead to renal failure. Telmisartan (TEL) is an approved angiotensin II type 1 receptor blocker for the treatment of hypertension and possesses nephroprotective efficacy. The study investigated the beneficial effect of TEL on renal oxidative stress, inflammatory response, and apoptosis in type 1 diabetic rats, pointing to the possible role of Nrf2/HO-1 signaling. Diabetes was induced by streptozotocin (STZ), and TEL (5 and 10 mg/kg) was supplement for 8 weeks. TEL ameliorated hyperglycemia, prevented body weight loss and kidney hypertrophy, decreased serum creatinine and urea, and prevented histopathological alterations in diabetic rats. Malondialdehyde (MDA), nitric oxide (NO), NF-κB p65 and TNF-α were increased, whereas GSH, SOD and Bcl-2 were decreased in the kidney of diabetic rats. Treatment with TEL ameliorated oxidative stress, suppressed NF-κB p65 and TNF-α, and boosted cellular antioxidant defenses and Bcl-2. TEL upregulated Nrf2 and HO-1 in the kidney of both normal and diabetic rats. In addition, TEL downregulated VEGF and MMP-9 in the kidney of diabetic rats. In silico molecular docking simulations revealed the potent binding affinity of TEL to NF-κB, MMP-9, Keap1 and HO-1. In conclusion, TEL attenuates DN by ameliorating hyperglycemia, oxidative stress, inflammation, apoptosis and angiogenesis and upregulation of Nrf2/HO-1 signaling.
Oxidative stress is an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control, and/or molecular damage altering cellular functions. This redox imbalance may trigger different responses depending on the antioxidant potential of a given cell, the level of reactive oxygen/nitrogen species (ROS/RNS) attained and the time of exposure, with protective effects being induced at low ROS/RNS levels in acute or short‐term conditions, and harmful effects after high ROS/RNS exposure in prolonged situations. Relevant conditions underlying liver redox imbalance include iron overload associated with ROS production via Fenton chemistry and the magnitude of the iron labile pool achieved, with low iron exposure inducing protective effects related to nuclear factor‐κB, signal transducer and activation of transcription 3, and nuclear factor erythroid‐related factor 2 (Nrf2) activation and upregulation of ferritin, hepcidin, acute‐phase response and antioxidant components, whereas high iron exposure causes drastic oxidation of biomolecules, mitochondrial dysfunction, and cell death due to necrosis, apoptosis and/or ferroptosis. Redox imbalance in nonalcoholic fatty liver disease (NAFLD) is related to polyunsaturated fatty acid depletion, lipogenic factor sterol regulatory element‐binding protein‐1c upregulation, fatty acid oxidation‐dependent peroxisome proliferator‐activated receptor‐α downregulation, low antioxidant factor Nrf2 and insulin resistance, a phenomenon that is exacerbated in nonalcoholic steatohepatitis triggering an inflammatory response. Thyroid hormone (T3) administration determines liver preconditioning against ischemia–reperfusion injury due to the redox activation of several transcription factors, AMP‐activated protein kinase, unfolded protein response and autophagy. High grade liver redox imbalance occurring in severe iron overload is adequately handled by iron chelation, however, that underlying NAFLD/NASH is currently under study in several Phase II and Phase III trials.
Capsanthin is the main carotenoid compound in red paprika (Capsicum annuum L.). However, little is known about the beneficial effects of capsanthin in nonalcoholic fatty liver disease (NAFLD). In this study, the hepatoprotective activity of capsanthin was investigated in a mouse model of NAFLD. Apolipoprotein-E knockout mice were fed with normal diet, Western-type diet (WD, NAFLD model), WD with capsanthin (0.5 mg/kg of body weight/day, CAP), WD with capsanthin-rich extract (25 mg/kg of body weight/day; CRE), or WD with red paprika powder (25 mg/kg of body weight/day, RPP) for 12 weeks. The carotenoid content in CRE or RPP was analyzed using ultraperformance liquid chromatography. The capsanthin concentration in CRE was 2067 mg/100 g of dry weight, which was 63% of total carotenoids. The oral administration of CRE or capsanthin significantly reduced the WD-induced increase in body weight and lipid accumulation in the liver (vs. the RPP group). In addition, CRE or capsanthin significantly inhibited the WD-induced increase in cholesterol and low-density lipoprotein levels. Furthermore, CRE or capsanthin showed reduced levels of plasma alanine and aspartate aminotransferase (ALT and AST, respectively), suggesting a steatohepatitis protective effect. Capsanthin regulated mRNA levels of peroxisome proliferator-activated receptor alpha (Pparα), carnitine palmitoyltransferase 1A (Cpt1a), acyl-CoA oxidase 1 (Acox1), and sterol regulatory element binding protein-1c (Srebp1c), which are associated with hepatic fatty acid metabolism. Overall, our results suggest that the capsanthin of red paprika plays a protective role against hepatic steatosis/steatohepatitis in NAFLD.
Non-alcoholic fatty liver disease (NAFLD) has a global prevalence of 25% and is a leading cause of cirrhosis and hepatocellular carcinoma. NAFLD encompasses a disease continuum from steatosis with or without mild inflammation (non-alcoholic fatty liver), to non-alcoholic steatohepatitis (NASH), which is characterised by necroinflammation and faster fibrosis progression than non-alcoholic fatty liver. NAFLD has a bidirectional association with components of the metabolic syndrome, and type 2 diabetes increases the risk of cirrhosis and related complications. Although the leading causes of death in people with NAFLD are cardiovascular disease and extrahepatic malignancy, advanced liver fibrosis is a key prognostic marker for liver-related outcomes and overall mortality, and can be assessed with combinations of non-invasive tests. Patients with cirrhosis should be screened for hepatocellular carcinoma and oesophageal varices. There is currently no approved therapy for NAFLD, although several drugs are in advanced stages of development. Because of the complex pathophysiology and substantial heterogeneity of disease phenotypes, combination treatment is likely to be required for many patients with NAFLD. Healthy lifestyle and weight reduction remain crucial to the prevention and treatment of NAFLD.
The prevalence of various hepatic diseases increases dramatically worldwide and regarded as a serious health problem. Sirtuins are one of the main strategic controllers of different cellular processes, including cell cycle, mitochondrial biogenesis, insulin secretion, redox balance, inflammation, and apoptosis. SIRT1 is the most prominent and broadly studied member of sirtuins that implicated in health status and longevity. Therefore, targeting the SIRT1 signaling pathways may be a reasonable therapeutic approach to treat different diseases, including hepatic disorders. Flavonoids are polyphenolic compounds widely present in different plants and possess beneficial effects against diverse diseases. In this review, we focused on the flavonoids, (−)-epicatechin, ampelopsin, baicalin, delphinidin, fisetin, epigallocatechin-3-gallate, luteolin, pinocembrin, quercetin, silibinin, trans-chalcone and xanthohumol, to verify whether their potential promising hepatoprotective effects are related to activation of SIRT1. Additionally, molecular modeling simulations were applied to explore the potential binding mode of these flavonoids to SIRT1. The complied information and molecular docking simulations suggested that SIRT1 signaling is involved in the beneficial pharmacologic activities of flavonoids in different hepatic diseases.
Aim:
Chronic inflammation links closely to insulin resistance and lipid metabolism in non-alcoholic fatty liver disease (NAFLD). Macrophage M1 activation plays an important role in the initiation and continuing of pro-inflammatory response of NAFLD. Our study was to investigate whether macrophage M1/M2 polarization switching would affect hepatic inflammation and lipid metabolism through modulation of PPAR-γ activity in vivo and in vitro.
Methods:
RAW264.7 macrophages were treated with different fatty acids and cell culture supernatants were collected to prepare conditioned media (CM). Different co-culture systems between primary hepatocytes and CM from macrophages were established. PPAR-γ agonist or antagonist was administered to regulate PPAR-γ activity and macrophage polarization. M1/M2 phenotype markers, inflammatory signaling pathway and lipid-related genes expression were determined. Wild-type C57BL/6 mice were fed a high-fat diet to induce NAFLD, and given rosiglitazone to regulate PPAR-γ activity in vivo.
Results:
Saturated fatty acids induced M1-polarized macrophages while polyunsaturated fatty acids induced M2-polarized macrophages. M1-polarized macrophages significantly promoted lipid synthesis and accumulation in primary hepatocytes through upregulation of toll-like receptor 4 (TLR4)/NF-κB signaling pathway. PPAR-γ agonist made lipid-induced M1-polarized macrophages switch to an M2-predominant phenotype, while PPAR-γ antagonist had the opposite effect. Macrophage polarization shifting subsequently affected lipid metabolism in primary hepatocytes. Administration of rosiglitazone improved high-fat diet induced hepatic steatosis and lipid metabolism through reducing hepatic TLR4/NF-κB expression and M1-polarized Kupffer cells.
Conclusions:
Lipid-induced macrophage M1 polarization promoted hepatic lipid metabolism. Modulation of PPAR-γ activity could shift macrophage polarization and subsequently affect lipid metabolism. Upregulation of the TLR4/NF-κB signaling pathway is closely linked to dysregulated lipid metabolism in NAFLD.
Significance:
Mitochondria represent a major source of intracellular reactive oxygen species (ROS) generation. This is often a consequence of oxidative phosphorylation (OXPHOS), which can produce ROS as a result of leakage from the electron transport chain (ETC). In addition, quality control mechanisms exist to protect cells at from cytotoxic ROS production. One such mechanism is selective autophagic degradation of ROS-producing mitochondria, termed mitophagy, that ultimately results in elimination of mitochondria in the lysosome. Recent Advances. However, while the relationship between mitophagy and ROS production are clearly interwoven, they have yet to be fully untangled. In some circumstances, mitochondrial ROS (mtROS) are elevated as as a consequence of mitophagy induction.
Critical issues:
In this review, we discuss mtROS generation and their detrimental effects on cellular viability. Additionally, we consider the cellular defense mechanisms that the eukaryotic cell employs to abrogate superfluous oxidative stress. In particular, we delve into the prominent mechanisms governing mitophagy induction that bear on oxidative stress.
Future directions:
Finally, we examine the pathological conditions associated with defective mitophagy where additional research may help to facilitate understanding.
Rumex dentatus L. is a flowering plant with promising therapeutic effects. This study
investigated the antioxidant efficacy of phenolic compounds isolated from R. dentatus L. in
vitro and by conducting density function theory (DFT) studies to explore the mechanisms of
action. The antioxidant, anti-inflammatory and antidiabetic effects of polyphenols-rich R.
dentatus extract (RDE) were investigated in type 2 diabetic rats. Phytochemical investigation of
the aerial parts of R. dentatus resulted in the isolation of one new and seven known compounds
isolated for the first time from this species. All isolated phenolics showed in vitro radical
scavenging activity. The antioxidant activity of the compounds could be oriented by the
hydrogen atom transfer and sequential proton loss electron transfer mechanisms in gas and
water phases, respectively. In diabetic rats, RDE attenuated hyperglycemia, insulin resistance
and liver injury and improved carbohydrate metabolism. RDE suppressed oxidative stress and
inflammation and upregulated PPARγ. In silico molecular docking analysis revealed the
binding affinity of the isolated compounds toward PPARγ. In conclusion, the computational
calculations were correlated with the in vitro antioxidant activity of R. dentatus derived
phenolics. R. dentatus attenuated hyperglycemia, liver injury, inflammation and oxidative
stress, improved carbohydrate metabolism and upregulated PPARγ in diabetic rats.
Fatty liver associated with metabolic dysfunction is common, affects a quarter of the population, and has no approved drug therapy. While pharmacotherapies are in development, response rates appear modest. The heterogeneous pathogenesis of metabolic fatty liver diseases and inaccuracies in terminology and definitions necessitate a reappraisal of nomenclature to inform clinical trial design and drug development. A group of experts sought to integrate current understanding of patient heterogeneity captured under the acronym nonalcoholic fatty liver disease (NAFLD) and provide suggestions on terminology that more accurately reflects pathogenesis and can help in patient stratification for management. Experts reached consensus that NAFLD does not reflect current knowledge and metabolic (dysfunction) associated fatty liver disease "MAFLD" was suggested as a more appropriate overarching term. This opens the door for efforts from the research community to update the nomenclature and sub-phenotype the disease in order to accelerate the translational path to new treatments.
Background & aims:
Although non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and NASH with advanced fibrosis are closely associated with type 2 diabetes mellitus (T2DM), their global prevalence rates have not been well described. Our aim was to estimate the prevalence of NAFLD, NASH, and advanced fibrosis among patients with T2DM, by regions of the world.
Methods:
We searched for terms including NAFLD, NASH and T2DM in studies published from January 1989 to September 2018, using PubMed, Ovid MEDLINE®, EMBASE and Web of Science. Strict exclusion criteria were applied. Regional and global mean prevalence weighted by population size in each country were estimated and pooled using random-effects meta-analysis. Potential sources of heterogeneity were investigated using stratified meta-analysis and meta-regression.
Results:
Among 80 studies from 20 countries that met our inclusion criteria, there were 49,419 individuals with T2DM (mean age 58.5 years, mean body mass index 27.9 kg/m2, and males 52.9%). The global prevalence of NAFLD among patients with T2DM was 55.5% (95% CI 47.3-63.7). Studies from Europe reported the highest prevalence (68.0% [62.1-73.0%]). Among 10 studies that estimated the prevalence of NASH, the global prevalence of NASH among individuals with T2DM was 37.3% (95% CI 24.7-50.0%). Seven studies estimated the prevalence of advanced fibrosis in patients with NAFLD and T2DM to be 17.0% (95% CI 7.2-34.8). Meta-regression models showed that geographic region and mean age (p <0.5) were associated with the prevalence of NAFLD, jointly accounting for 63.9% of the heterogeneity.
Conclusions:
This study provides the global prevalence rates for NAFLD, NASH, and advanced fibrosis in patients with T2DM. These data can be used to estimate the clinical and economic burden of NASH in patients with T2DM around the world.
Lay summary:
Non-alcoholic fatty liver disease (NAFLD) is now recognized as the most prevalent chronic liver disease worldwide. Type 2 diabetes mellitus (T2DM) is an important risk factor for NAFLD. Additionally, T2DM seems to accelerate the progression of liver disease in NAFLD. Despite the high prevalence and serious clinical implications of NAFLD in patients with T2DM, it is usually overlooked in clinical practice. This meta-analysis provides evidence of the high prevalence of NAFLD and NASH in patients with T2DM. In this context, increasing awareness about the importance of NAFLD in patients with T2DM among all important stakeholders (primary care physicians, specialists, and health policy makers) must be prioritized.
The obesity epidemic is closely associated with the rising prevalence and severity of nonalcoholic fatty liver disease (NAFLD): obesity has been linked not only with simple steatosis (SS), but also with advanced disease, i.e., nonalcoholic steatohepatitis (NASH), NASH-related cirrhosis and hepatocellular carcinoma. As a consequence, apart from increasing all-cause mortality, obesity seems to increase liver-specific mortality in NAFLD patients. Given the lack of approved pharmacological interventions for NAFLD, targeting obesity is a rational option for its management. As the first step, lifestyle modification (diet and exercise) is recommended, although it is difficult to achieve and sustain. When the first step fails, adding pharmacotherapy is recommended. Several anti-obesity medications have been investigated in NAFLD (e.g., orlistat, glucagon-like peptide-1 analogs), other anti-obesity medications have not been investigated (e.g., lorcaserin, phentermine hydrochloric, phentermine/topiramate and naltrexone/bupropion), whereas some medications with weight-lowering efficacy have not been approved for obesity (e.g., sodium-glucose cotransporter-2 inhibitors, farnesoid X receptor ligands). If the combination of lifestyle modification and pharmacotherapy also fails, then bariatric surgery should be considered in selected morbidly obese individuals. This review summarizes best evidence linking obesity with NAFLD and presents related therapeutic options.
Enhanced iron levels in liver are associated with oxidative stress development and damage with increased fat accumulation. The aim of this work was to assess the hypothesis that antioxidant-rich extra virgin olive oil (AR-EVOO) counteracts iron-rich diet (IRD)-induced oxidative stress hindering hepatic steatosis. Male Wistar rats were fed and IRD (200mg iron/kg diet) versus a control diet (CD; 50mg iron/kg diet) with alternate AR-EVOO supplementation (100mg/day) for 21 days. IRD induced liver steatosis and oxidative stress (higher levels of protein oxidation and lipid peroxidation with glutathione depletion), mitochondrial dysfunction (decreased citrate synthase and complex I and II activities) and loss of polyunsaturated fatty acids (PUFAs), with a drastic enhancement in the sterol regulatory element-binding protein-1c (SREBP-1c)/peroxisome proliferator-activated receptor-α (PPAR-α) ratio upregulating the expression of lipogenic enzymes (acetyl-CoA carboxylase, fatty acid (FA) synthase and stearoyl desaturase 2) and downregulating those involved in FA oxidation (carnitine palmitoyl transferase and acyl-CoA oxidase) over values in the CD group. IRD also upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and its target genes. AR-EVOO supplementation alone did not modify the studied parameters, however, IRD combined with AR-EVOO administration returned IRD-induced changes to baseline levels of the CD group. It is concluded that IRD-induced non-alcoholic fatty liver disease (NAFLD) is prevented by AR-EVOO supplementation, which might be related to the protective effects of its components such as hydroxytyrosol, oleic acid, tocopherols and/or PUFAs, thus representing a suitable anti-steatotic strategy to avoid progression into more severe stages of the disease, underlying NAFLD associated with iron overloading pathologies or obesity.
There has been a rise in the prevalence of nonalcoholic fatty liver disease (NAFLD), paralleling a worldwide increase in diabetes and metabolic syndrome. NAFLD, a continuum of liver abnormalities from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), has a variable course but can lead to cirrhosis and liver cancer. Here we review the pathogenic and clinical features of NAFLD, its major comorbidities, clinical progression and risk of complications and in vitro and animal models of NAFLD enabling refinement of therapeutic targets that can accelerate drug development. We also discuss evolving principles of clinical trial design to evaluate drug efficacy and the emerging targets for drug development that involve either single agents or combination therapies intended to arrest or reverse disease progression.
NAFLD is one of the most important causes of liver disease worldwide and will probably emerge as the leading cause of end-stage liver disease in the coming decades, with the disease affecting both adults and children. The epidemiology and demographic characteristics of NAFLD vary worldwide, usually parallel to the prevalence of obesity, but a substantial proportion of patients are lean. The large number of patients with NAFLD with potential for progressive liver disease creates challenges for screening, as the diagnosis of NASH necessitates invasive liver biopsy. Furthermore, individuals with NAFLD have a high frequency of metabolic comorbidities and could place a growing strain on health-care systems from their need for management. While awaiting the development effective therapies, this disease warrants the attention of primary care physicians, specialists and health policy makers.
Introduction: Oxidative stress is central to the pathogenesis of non-alcoholic steatohepatitis. The reactive oxygen species (ROS) that characterise oxidative stress are generated in several cellular sites and their production is influence by multi-organ interactions.
Areas covered: Mitochondrial dysfunction is the main source of ROS in fatty liver and is closely related to endoplasmic reticulum stress. Both are caused by lipotoxicity and together these three factors form a cycle of progressive organelle damage, resulting in sterile inflammation and apoptosis. Adipose tissue inflammation and intestinal dysbiosis provide substrates for ROS formation and trigger immune activation. Obstructive sleep apnea and abnormal divalent metal metabolism may also play a role.
Expert commentary: The majority of available high-quality data originates from studies in adults and there are fewer therapeutic trials performed in pediatric cohorts, therefore conclusions are generalised to children. Establishing the role of organelle interactions, and its relationship with oxidative stress in steatohepatitis, is a rapidly evolving area of research.
Umbelliferone (UMB) is a coumarin derivative with promising hepatoprotective effects. In this study, we examined the possible protective effects of UMB against cyclophosphamide (CP)-induced hepatotoxicity, addressing the question of the possible role of nuclear factor erythroid 2-related factor 2 (Nrf2) and peroxisome proliferator activated receptor gamma (PPARγ). Wistar rats were orally administered UMB at doses 50 and 100 mg/kg two weeks prior to CP injection. Five days after CP administration, the rats were sacrificed and samples were collected for analyses. CP induced a significant increase in circulating liver marker enzymes and pro-inflammatory cytokines. Hepatic lipid peroxidation and nitric oxide levels, and nuclear factor-kappaB (NF-κB) and inducible nitric oxide synthase (iNOS) expression were significantly increased following CP administration. UMB supplementation attenuated CP-induced inflammation and oxidative stress as assessed by restoration of the activity and expression of the antioxidant defenses, and suppression of pro-inflammatory cytokines. Histological examination also showed that UMB could significantly reduce CP-induced alterations. CP-induced rats showed significant down-regulation of Nrf2, HO-1 and PPARγ, an effect that was markedly reversed by UMB. In conclusion, the hepatoprotective effects of UMB appear to depend on co-activation of PPARγ and Nrf2, and subsequent suppression of oxidative stress and inflammation.
Betulin is extracted from birch tree bark and exerts diverse pharmacological activities. The present study was designed to investigate the protective effect of betulin (BE) on cognitive decline in streptozotocin (STZ)-induced diabetic rats. The diabetic model was built by streptozotocin (STZ) (30mg/kg, ip). After 4 weeks, the diabetic rats were treated with vehicle or BE (20mg/kg, 40mg/kg) for 4 weeks. The oral glucose tolerance (OGTT) and serum insulin were detected. Three days later, Morris water maze (MWM) test were used to evaluate memory function. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in hippocampus were examined. Inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in serum and hippocampus were measured. The protein expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NF-κB pathways-related molecules in hippocampus were examined. As a results, BE could improve glucose intolerance and modify basal learning performance. Treatment with BE significantly restored SOD activity and decreased MDA content in hippocampus. BE also markedly reduced the contents of inflammatory cytokines in serum and hippocampus. Furthermore, administration of BE effectively upregulated the expressions of Nrf2, HO-1 and blocked the phosphorylations of IκB, NF-κB. In summary, BE might exhibit protective effect on cognitive decline in STZ-induced diabetic rats through HO-1/Nrf-2/NF-κB pathway.
The present study was conducted to investigate the protective effect of betulin, a triterpene from the bark of Betula platyphylla Suk, against ethanol-induced alcoholic liver injury and its possible underlying mechanisms. In vitro, human hepatic stellate cell line, LX-2 cells were treated with betulin (6.25, 12.5 and 25 μM) prior to ethanol (50 mM) for 24 h. Cell viability was analyzed by methyl thiazolyl tetrazolium assay, protein expressions were assessed by Western blot. In vivo, we induced alcoholic liver injury in male C57BL/6 mice, placing them on Lieber-DeCarli ethanol-containing diets for 10 days and then administering a single dose of ethanol (5 g/kg body weight) via gavage. Betulin (20 and 50 mg/kg) were given by gavage every day. In vitro results showed that betulin effectively decreased LX-2 cell viability, attenuated collagen-I, α-smooth muscle actin (α-SMA) levels, activated liver kinase B-1 (LKB1) and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. Betulin suppressed the expression of sterol regulatory element-binding protein-1 (SREBP-1), and genetic deletion of AMPK blocked the effect of betulin on SREBP-1 in ethanol treated LX-2 cells. In vivo, betulin attenuated the increases in serum aminotransferase and triglyceride levels in the mice fed with chronic-binge ethanol, while significantly inhibited SREBP-1 expression and activated LKB1-AMPK phosphorylation. Additionally, betulin enhanced the sirtuin 1 (SIRT1) expression mediated by ethanol. Taken together, betulin alleviates alcoholic liver injury possibly through blocking the regulation of SREBP-1 on fatty acid synthesis and activating SIRT1-LKB1-AMPK signaling pathway.
Background and aim:
NAFLD is a major cause of liver disease worldwide. We estimated the global prevalence, incidence, progression and outcomes of NAFLD and NASH.
Methods:
Pubmed/MEDLINE were searched from 1989-2015 for terms involving epidemiology and progression of NAFLD.
Exclusions:
selected groups (only morbidly obese or diabetics or pediatric), no data on alcohol consumption or other liver diseases. Incidence of HCC, cirrhosis, overall mortality and liver-related mortality were determined. NASH required histologic criteria. All studies were reviewed by 3 independent investigators. Analysis was stratified by region, diagnostic technique, biopsy indication and study population. We used random-effects models to provide point estimates (95% CI) of prevalence, incidence, mortality and incidence rate ratios, and meta-regression with sub-group analysis to account for heterogeneity.
Results:
Out of 729 studies, 86 were included with a sample size of 8,515,431 from 22 countries. Global prevalence of NAFLD is 25.24% (22.10-28.65) with highest prevalence in Middle East and South America and lowest in Africa. Metabolic comorbidities associated with NAFLD included obesity [51.34% (41.38-61.20)], type 2 diabetes [22.51% (17.92-27.89)], hyperlipidemia [69.16% (49.91-83.46%)], hypertension [(39.34% (33.15-45.88)]] and metabolic syndrome [42.54% (30.06-56.05)] . Fibrosis progression proportion, measured in Brunt's score, and mean annual rate of progression in NASH were 40.76% (34.69-47.13) and 0.09 (0.06-0.12). HCC incidence among NAFLD patients was 0.44/1000 person-years (0.29-0.66). Liver-specific mortality and overall mortality among NAFLD and NASH were 0.77/1000 person-years (0.33-1.77) and 11.77/1000 person-years (7.10-19.53), 15.44/1000 person-years (11.72-20.34) and 25.56/1000 person-years (6.29-103.80). Incidence Risk Ratios for liver-specific and overall mortality for NAFLD were 1.94 (1.28-2.92) and 1.05 (0.70-1.56).
Conclusions:
As the global epidemic of obesity fuels metabolic conditions, the clinical and economic burden of NAFLD will become enormous.f This article is protected by copyright. All rights reserved.
Generation of reactive oxygen species (ROS) in response to fatty acids accumulation has been classically proposed as a possible "second hit" triggering progression from simple steatosis to non-alcoholic steatohepatitis (NASH). In this study we challenged hepatocyte-specific Keap1 knockout mice (Keap1(Δhepa)) and littermate Cre- controls (Keap1(fx/fx)) with two different diet models of NASH in order to evaluate the effects of the anti-oxidant transcription factor Nrf2 over-activation on hepatic metabolism and disease progression. After 4 weeks of MCD diet the liver/body weight ratio of Keap1(Δhepa) mice was significantly higher compared to littermate controls with no differences in total body weight. Strikingly, liver histology revealed a dramatic reduction of lipid droplets confirmed by a decreased content of intra-hepatic triglycerides in Keap1(Δhepa) compared to controls. In parallel to reduced expression of genes involved in lipid droplet formation, protein expression of Liver X Receptor (LXRα/β) and Peroxisome proliferator-activated receptor α (PPARα) was significantly decreased. In contrast, genes involved in mitochondrial lipid catabolism were markedly up-regulated in Keap1(Δhepa) livers. A similar phenotype characterized by inhibition of lipogenesis in favor of increased mitochondrial catabolic activity was also observed after 13 weeks of western diet administration. MCD-induced apoptosis was significantly dampened in Keap1(Δhepa) compared to Keap1(fx/fx) as detected by TUNEL, cleaved caspase-3 and Bcl-2 protein expression analyses. However, no differences in inflammatory F4/80- and CD11b-positive cells and pro-fibrogenic genes were detected between the two groups. Although hepatic lack of Keap1 did not ameliorate inflammation, the resulting constitutive Nrf2 over-activation in hepatocytes strongly reduced hepatic steatosis via enhanced lipid catabolism and repressed de novo lipogenesis during murine NASH development.
Objective:
Resveratrol (RSV) is the most studied natural compound that activates sirtuins, which produce beneficial metabolic effects on lipid and glucose metabolism. The aim of the present study was to investigate the role of resveratrol in preventing nonalcoholic fatty liver disease (NAFLD) and expression of liver inflammatory markers in mice treated with a high-fat diet.
Methods and procedures:
Eighteen male mice were divided into three groups and fed for 60 d with a standard diet (ST), high-fat diet (HFD), or high-fat diet plus resveratrol (HFD + RSV, 30 mg/kg/d). Body weight, food intake, and serum total cholesterol, triacylglycerol, insulin, alanine transaminase (ALT), and aspartate aminotransferase (AST) were evaluated. Liver histology was analyzed. Expression of ACC, PPAR-γ, ChREBP, SREBP-1 c, CPT-1, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), NF-κB, interleukin 1 β (IL-1 β), and SIRT1 were evaluated by quantitative real-time reverse transcriptase PCR (qRT-PCR).
Results:
The major finding of the present study was that RSV reduced body fat, total cholesterol, triacylglycerol, transaminases, and insulin plasma level. These results were accompanied with a significant reduction in TNF-α, IL-6, and NF-κB mRNA expression in the liver. Analyses of liver adipogenesis related genes indicated that ACC, PPAR-γ, and SREBP-1 mRNA expression were significantly suppressed in HFD + RSV mice. In addition, we observed increased expression of SIRT1 in the HFD + RSV group.
Conclusions:
We observed that treatment with resveratrol improved lipid metabolism, and decreased NAFLD and pro-inflammatory profile in liver of mice with obesity-inducible diets. These data suggest an important clinical application of RSV in preventing liver diseases.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
The autoxidation of pyrogallol was investigated in the presence of EDTA in the pH range 7.9–10.6.
The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99% by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, O2·−, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O2·− -independent mechanisms rapidly become dominant.
Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H2O2 is the stable product of oxygen and that H2O2 is not involved in the autoxidation mechanism.
A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol.
A plausible explanation is given for the non-competitive part of the inhibition of catechol O-methyltransferase brought about by pyrogallol.
Unlabelled:
Previous studies have shown familial aggregation of insulin resistance and nonalcoholic fatty liver disease (NAFLD). Therefore, we aimed to examine whether family history of diabetes mellitus (DM) is associated with nonalcoholic steatohepatitis (NASH) and fibrosis in patients with NAFLD. This was a cross-sectional analysis in participants of the NAFLD Database study and PIVENS trial who had available data on family history of DM. One thousand and sixty-nine patients (63% women), with mean age of 49.6 (± 11.8) years and body mass index (BMI) of 34.2 (± 6.4) kg/m(2) , were included. Thirty percent had DM, and 56% had a family history of DM. Both personal history of DM and family history of DM were significantly associated with NASH, with an odds ratio (OR) of 1.93 (95% confidence interval [CI]: 1.37-2.73; P <0.001) and 1.48 (95% CI: 1.11-1.97; P = 0.01) and any fibrosis with an OR of 3.31 (95% CI: 2.26-4.85; P < 0.001) and 1.66 (95% CI: 1.25-2.20; P < 0.001), respectively. When the models were adjusted for age, sex, BMI, ethnicity, and metabolic traits, the association between diabetes and family history of DM with NASH showed an increased adjusted OR of 1.76 (95% CI: 1.13-2.72; P < 0.001) and 1.34 (95% CI: 0.99-1.81; P = 0.06), respectively, and with any fibrosis with a significant adjusted OR of 2.57 (95% CI: 1.61-4.11; P < 0.0001) and 1.38 (95% CI: 1.02-1.87; P = 0.04), respectively. After excluding patients with personal history of diabetes, family history of DM was significantly associated with the presence of NASH and any fibrosis with an adjusted OR of 1.51 (95% CI: 1.01-2.25; P = 0.04) and 1.49 (95% CI: 1.01-2.20; P = 0.04), respectively.
Conclusions:
Diabetes is strongly associated with risk of NASH, fibrosis, and advanced fibrosis. Family history of diabetes, especially among nondiabetics, is associated with NASH and fibrosis in NAFLD.
Nonalcoholic fatty liver disease (NAFLD) is a burgeoning health problem that affects one-third of adults and an increasing
number of children in developed countries. The disease begins with the aberrant accumulation of triglyceride in the liver,
which in some individuals elicits an inflammatory response that can progress to cirrhosis and liver cancer. Although NAFLD
is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood, and therapeutic options
are limited. Here, we discuss recent mechanistic insights into NAFLD, focusing primarily on those that have emerged from human
genetic and metabolic studies.
Sterol regulatory element-binding proteins (SREBPs) are major transcription factors activating the expression of genes involved in biosynthesis of cholesterol, fatty acid and triglyceride. In this study, we identified a small molecule, betulin, that specifically inhibited the maturation of SREBP by inducing interaction of SREBP cleavage activating protein (SCAP) and Insig. Inhibition of SREBP by betulin decreased the biosynthesis of cholesterol and fatty acid. In vivo, betulin ameliorated diet-induced obesity, decreased the lipid contents in serum and tissues, and increased insulin sensitivity. Furthermore, betulin reduced the size and improved the stability of atherosclerotic plaques. Our study demonstrates that inhibition SREBP pathway can be employed as a therapeutic strategy to treat metabolic diseases including type II diabetes and atherosclerosis. Betulin, which is abundant in birch bark, could be a leading compound for development of drugs for hyperlipidemia.
SIRT1, a homolog of yeast Sir2, is a type III NAD(+) dependent histone and protein deacetylase. Previous studies of mice carrying liver specific deletion of exon 4 of the Sirt1 gene revealed opposite responses of mutant mice to a high-fat diet in terms of fatty liver formation, which obscures the function of SRIT1 in liver development and lipid metabolism. To investigate this, we deleted exons 5 and 6 of Sirt1 in the liver by using a Cre-loxP approach. Western blot using an antibody to N-terminal SIRT1 does not detect a truncated protein in the liver of the mutant mice (Sirt1(flox5-6/flox5-6);Alb-Cre), suggesting a null mutation for SIRT1 is generated in the liver. Unlike the previously reported phenotypes, the Sirt1(flox5-6/flox5-6);Alb-Cre mice develop fatty liver under a normal feeding condition. The disease starts at two months of age and incidence increases as the animals become older, affecting 78% of them when they are over one year of age. We showed that the steatosis is accompanied by altered expression of a number of genes, including increased expression of ChREBP, which acts as one of the central determinants of lipid synthesis in the liver. This data uncovers an important role of SIRT1 in regulating lipid metabolism in the liver, and the SIRT1 mutant mice may serve as an animal model for studying human fatty liver disease and facilitate the development of effective therapeutic approach for the disease.
Oxidative stress is a critical mediator in liver injury of steatohepatitis. The transcription factor Nrf2 serves as a cellular stress sensor and is a key regulator for induction of hepatic detoxification and antioxidative stress systems. The involvement of Nrf2 in defense against the development of steatohepatitis remains unknown. We aimed to investigate the protective roles of Nrf2 in nutritional steatohepatitis using wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. WT and Nrf2-null mice were fed a methionine- and choline-deficient (MCD) diet for 3 and 6 wk, and the liver tissues were analyzed for pathology and for expression levels of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. In WT mice fed an MCD diet, Nrf2 was potently activated in the livers, and steatohepatitis did not develop over the observation periods. However, in Nrf2-null mice fed an MCD diet, the pathological state of the steatohepatitis was aggravated in terms of fatty changes, inflammation, fibrosis, and iron accumulation. In the livers of the Nrf2-null mice, oxidative stress was significantly increased compared with that of WT mice based on the increased levels of 4-hydroxy-2-nonenal and malondialdehyde. This change was associated with the decreased levels of glutathione, detoxifying enzymes, catalase, and superoxide dismutase activity. Correlating well with the liver pathology, the mRNA levels of factors involved in fatty acid metabolism, inflammatory cytokines, and fibrogenesis-related genes were significantly increased in the livers of the Nrf2-null mice. These findings demonstrate that Nrf2 deletion in mice leads to rapid onset and progression of nutritional steatohepatitis induced by an MCD diet. Activation of Nrf2 could be a promising target toward developing new options for prevention and treatment of steatohepatitis.
Hepatic metabolic derangements are key components in the development of fatty liver, insulin resistance, and atherosclerosis. SIRT1, a NAD+-dependent protein deacetylase, is an important regulator of energy homeostasis in response to nutrient availability. Here we demonstrate that hepatic SIRT1 regulates lipid homeostasis by positively regulating peroxisome proliferators-activated receptor alpha (PPARalpha), a nuclear receptor that mediates the adaptive response to fasting and starvation. Hepatocyte-specific deletion of SIRT1 impairs PPARalpha signaling and decreases fatty acid beta-oxidation, whereas overexpression of SIRT1 induces the expression of PPARalpha targets. SIRT1 interacts with PPARalpha and is required to activate PPARalpha coactivator PGC-1alpha. When challenged with a high-fat diet, liver-specific SIRT1 knockout mice develop hepatic steatosis, hepatic inflammation, and endoplasmic reticulum stress. Taken together, our data indicate that SIRT1 plays a vital role in the regulation of hepatic lipid homeostasis and that pharmacological activation of SIRT1 may be important for the prevention of obesity-associated metabolic diseases.
The NAD(+)-dependent deacetylase SIRT1 controls metabolic processes in response to low nutrient availability. We report the metabolic phenotype of mice treated with SRT1720, a specific and potent synthetic activator of SIRT1 that is devoid of direct action on AMPK. SRT1720 administration robustly enhances endurance running performance and strongly protects from diet-induced obesity and insulin resistance by enhancing oxidative metabolism in skeletal muscle, liver, and brown adipose tissue. These metabolic effects of SRT1720 are mediated by the induction of a genetic network controlling fatty acid oxidation through a multifaceted mechanism that involves the direct deacetylation of PGC-1alpha, FOXO1, and p53 and the indirect stimulation of AMPK signaling through a global metabolic adaptation mimicking low energy levels. Combined with our previous work on resveratrol, the current study further validates SIRT1 as a target for the treatment of metabolic disorders and characterizes the mechanisms underlying the therapeutic potential of SIRT1 activation.
Alcoholic fatty liver is a potentially pathologic condition which can progress to steatohepatitis, fibrosis, and cirrhosis if alcohol consumption is continued. Alcohol exposure may induce fatty liver by increasing NADH/NAD(+) ratio, increasing sterol regulatory element-binding protein-1 (SREBP-1) activity, decreasing peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activity, and increasing complement C3 hepatic levels. Alcohol may increase SREBP-1 activity by decreasing the activities of AMP-activated protein kinase and sirtuin-1. Tumor necrosis factor-alpha (TNF-alpha) produced in response to alcohol exposure may cause fatty liver by up-regulating SREBP-1 activity, whereas betaine and pioglitazone may attenuate fatty liver by down-regulating SREBP-1 activity. PPAR-alpha agonists have potentials to attenuate alcoholic fatty liver. Adiponectin and interleukin-6 may attenuate alcoholic fatty liver by up-regulating PPAR-alpha and insulin signaling pathways while down-regulating SREBP-1 activity and suppressing TNF-alpha production. Recent studies show that paracrine activation of hepatic cannabinoid receptor 1 by hepatic stellate cell-derived endocannabinoids also contributes to the development of alcoholic fatty liver. Furthermore, oxidative modifications and inactivation of the enzymes involved in the mitochondrial and/or peroxisomal beta-oxidation of fatty acids could contribute to fat accumulation in the liver.