Dysregulation of the unfolded protein response in db/db mice with diet-induced steatohepatitis.
ABSTRACT: In humans with nonalcoholic fatty liver, diabetes is associated with more advanced disease. We have previously shown that diabetic db/db mice are highly susceptible to methionine choline-deficient diet (MCD)-induced hepatic injury. Because activation of the unfolded protein response (UPR) is an important adaptive cellular mechanism in diabetes, obesity, and fatty liver, we hypothesized that dysregulation of the UPR may partially explain how diabetes could promote liver injury. Db/db and db/m mice were fed the MCD or control diet for 4 weeks to characterize differences in UPR activation and downstream injury. Wildtype mice (C57BLKS/J) fed the MCD or control diet were treated with SP600125; a c-Jun N-terminal kinase (JNK) inhibitor and its effect on liver injury and UPR activation was measured. The MCD diet resulted in global up-regulation of the UPR in both diabetic db/db and nondiabetic db/m mice. db/db mice had an inadequate activation of recovery pathways (GADD34, XBP-1(s)) and accentuated activation of injury pathways related to persistent eif2-? phosphorylation (activating transcription factor 4 [ATF-4], C/EBP homologous transcription factor [CHOP], oxireductase endoplasmic reticulum oxidoreductin-1 [ERO-1?], JNK, nuclear factor kappaB [NF-?B]) compared to db/m mice. This led to increased expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-?), ICAM-1, and MCP-1 compared to db/m mice. Interestingly, whereas pharmacologic JNK inhibition did not prevent the development of MCD diet-induced steatohepatitis, it did attenuate UPR and downstream inflammatory signaling.MCD-fed db/db mice develop a more proinflammatory milieu than db/m mice associated with an impaired ability to dephosphorylate eif2-? through GADD34, impairing cellular recovery. These data may enhance our understanding of why diabetics with nonalcoholic steatohepatitis are prone to develop more severe liver injury than nondiabetic patients.
Project description:Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in the Western population. By mechanisms that are not completely understood, this disease may progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). db/db mice spontaneously develop hepatic steatosis, which progresses to NASH when these mice are fed a methionine choline-deficient (MCD) diet. The goal of our studies was to identify lipid and methionine metabolism pathways affected by MCD feeding to determine potential causal events leading to the development of NASH from benign steatosis. db/db mice fed the MCD diet for 2 weeks exhibited signs of incipient NASH development such as upregulated cytokines and chemokines. At this time point, MCD diet feeding caused S-adenosylmethionine (SAMe) depletion in db/db mice, while wild-type mice on the same diet retained hepatic SAMe levels. SAMe depletion exerts pleiotropic effects upon liver homeostasis and is commonly associated with a variety of liver insults such as thioacetamide, CCL4, and alcohol treatment; thus, SAMe depletion may serve as the second hit in NASH development. It is possible that differences in hepatic lipid and/or methionine metabolism between wild-type and db/db mice underlay the differential maintenance of SAMe levels during methionine and choline restriction. Indeed, db/db mice exhibited inhibited lipid oxidation pathways, which may be a priming factor for NASH development, and db/db mice fed the MCD diet had differential methionine adenosyltransferase (MAT) expression. The occurrence of SAMe depletion at this early, benign stage of NASH development in db/db mice with fatty liver suggests that SAMe supplementation may be (A) targeted to individuals susceptible to NASH (i.e., NAFLD patients) and (B) preventative of NASH before substantial liver injury has occurred.
Project description:Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and type 2 diabetes and is characterized by the accumulation of fat in the liver (steatosis). NAFLD can transition into non-alcoholic steatohepatitis (NASH), with liver cell injury, inflammation, and an increased risk of fibrosis. We previously found that injections of either 1866, a synthetic ligand for the lectin receptor CD209, or DANA, a sialidase inhibitor, can inhibit inflammation and fibrosis in multiple animal models. The methionine and choline-deficient (MCD) diet is a model of NASH which results in the rapid induction of liver steatosis and inflammation. In this report, we show that for C57BL/6 mice on a MCD diet, injections of both 1866 and DANA reversed MCD diet-induced decreases in white fat, decreases in adipocyte size, and white fat inflammation. However, these effects were not observed in type 2 diabetic db/db mice on a MCD diet. In db/db mice on a MCD diet, 1866 decreased liver steatosis, but these effects were not observed in C57BL/6 mice. There was no correlation between the ability of 1866 or DANA to affect steatosis and the effects of these compounds on the density of liver macrophage cells expressing CLEC4F, CD64, F4/80, or Mac2. Together these results indicate that 1866 and DANA modulate adipocyte size and adipose tissue macrophage populations, that 1866 could be useful for modulating steatosis, and that changes in the local density of 4 different liver macrophages cell types do not correlate with effects on liver steatosis.
Project description:Hepatic aldose reductase (AR) expression is known to be induced in liver diseases, including hepatitis and hepatocellular carcinoma. However, the role of AR in the development of these diseases remains unclear. We performed this current study to determine whether and how AR might be involved in the development of diet-induced nonalcoholic steatohepatitis. Our results showed that the level of AR protein expression was significantly higher in db/db mice fed the methionine-choline-deficient (MCD) diet than in mice fed the control diet. In parallel with the elevation in AR, steatohepatitis was observed in MCD diet-fed mice, and this diet-induced steatohepatitis was significantly attenuated by lentiviral-mediated knock-down of the AR gene. This suppressive effect of AR knock-down was associated with repressed levels of serum alanine aminotransferase and hepatic lipoperoxides, reduced mRNA and protein expression of hepatic cytochrome P450 2E1 (CYP2E1), and decreased mRNA expression of pro-inflammatory tumor necrosis factor-? (TNF-?) and interleukin-6 (IL-6). Moreover, AR-induced elevations on the level of CYP2E1 expression, reactive oxygen species, mRNA expression of TNF-? and IL-6 were confirmed in AML12 hepatocytes. Further, lentiviral-mediated knock-down of AR ameliorated MCD diet-induced collagen deposition in the livers of db/db mice. With the improvement in liver fibrosis, the mRNA levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-2 (MMP-2), two genes involved in hepatic fibrogenesis, were found to be significantly suppressed, while TIMP-2 and MMP-13 were unaffected. Together these data indicate that inhibition of AR alleviates the MCD diet-induced liver inflammation and fibrosis in db/db mice, probably through dampening CYP2E1 mediated-oxidative stress and ameliorating the expression of pro-inflammatory cytokines.
Project description:Hyperhomocysteinemia has been correlated with hepatic steatosis and activation of the unfolded protein response (UPR), yet a causal relationship has not been established. Although methionine and choline are essential components of homocysteine metabolism, the role of homocysteine in the pathogenesis of a methionine- and choline-deficient (MCD) diet remains unknown. We explored the effects of homocysteine supplementation on hepatic steatosis and the UPR in mice fed a control or MCD diet. Mice fed the MCD diet developed severe hyperhomocysteinemia and activation of the hepatic UPR. Supplementing the MCD diet with homocysteine attenuated the MCD diet-induced hepatic UPR activation and other injurious effects of the MCD diet including hepatic cholesterol accumulation, weight loss, and plasma ALT elevation. Homocysteine supplementation replenished the MCD diet-induced depletion of hepatic S-adenosylmethionine (SAM). Depleting SAM in HepG2 cells using MAT1alpha siRNA or cycloleucine resulted in enhanced activation of the UPR upon exposure to thapsigargin. Mice fed a control diet supplemented with homocysteine had a 3-fold elevation in plasma homocysteine level by 2 weeks and 6-fold elevation by 6 weeks but demonstrated no other pathophysiologic change. In summary, we found that homocysteine attenuates MCD diet-induced hepatic UPR activation, likely via repletion of hepatic SAM. Furthermore, homocysteine supplementation alone does not cause hepatic steatosis or UPR activation despite inducing hyperhomocysteinemia. These studies indicate that although hyperhomocysteinemia is often associated with hepatic steatosis and UPR activation, these effects may be a secondary response rather than a direct effect of homocysteine.
Project description:Nonalcoholic fatty liver disease is a prevalent form of chronic liver disease that can progress to the more advanced stage of nonalcoholic steatohepatitis (NASH). NASH has been shown to alter drug transporter regulation and may have implications in the development of adverse drug reactions. Several experimental rodent models have been proposed for the study of NASH, but no single model fully recapitulates all aspects of the human disease. The purpose of the current study was to determine which experimental NASH model best reflects the known alterations in human drug transporter expression to enable more accurate drug disposition predictions in NASH. Both rat and mouse NASH models were used in this investigation and include the methionine and choline deficient (MCD) diet model, atherogenic diet model, ob/ob and db/db mice, and fa/fa rats. Pathologic scoring evaluations demonstrated that MCD and atherogenic rats, as well as ob/ob and db/db mice, developed NASH. Liver mRNA and protein expression analyses of drug transporters showed that in general, efflux transporters were induced and uptake transporters were repressed in the rat MCD and the mouse ob/ob and db/db models. Lastly, concordance analyses suggest that both the mouse and rat MCD models as well as mouse ob/ob and db/db NASH models show the most similarity to human transporter mRNA and protein expression. These results suggest that the MCD rat and mouse model, as well as the ob/ob and db/db mouse models, may be useful for predicting altered disposition of drugs with similar kinetics across humans and rodents.
Project description:The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing in parallel with the prevalence of obesity. DNA damage-inducible protein 34 (GADD34/Ppp1r15a), originally isolated from UV-inducible transcripts in Chinese hamster ovary (CHO) cells, dephosphorylates several kinases that function in important signaling cascades, including dephosphorylation of eIF2?. We examined the effects of GADD34 on natural life span by using GADD34-deficient mice. Here we observed for the first time that with age GADD34-deficient mice become obese, developing fatty liver followed by liver cirrhosis, hepatocellular carcinoma, and insulin resistance. We found that myofibroblasts and immune cells infiltrated the portal veins of aged GADD34-deficient mouse livers. A high-fat diet (HFD) induced a higher level of steatosis in young GADD34-deficient mice compared with WT mice. Differentiation into fat is dependent on insulin signaling. Insulin signaling in young GADD34-deficient mice was higher than that in WT mice, which explained the higher fat differentiation of mouse embryonic fibroblasts (MEFs) observed in GADD34-deficient mice. Through aging or a HFD, insulin signaling in GADD34-deficient liver converted to be down regulated compared with WT mice. We found that a HFD or palmitate treatment converted insulin signaling by up-regulating TNF-? and JNK.
Project description:Fatty liver is associated with endoplasmic reticulum stress and activation of the hepatic unfolded protein response (UPR). Reduced hepatic expression of the UPR regulator X-box binding protein 1 spliced (XBP1s) is associated with human nonalcoholic steatohepatitis (NASH), and feeding mice a high-fat diet with fructose/sucrose causes progressive, fibrosing steatohepatitis. This study examines the role of XBP1 in nonalcoholic fatty liver injury and fatty acid-induced cell injury. Hepatocyte-specific Xbp1-deficient (Xbp1(-/-)) mice were fed a high-fat/sugar (HFS) diet for up to 16 wk. HFS-fed Xbp1(-/-) mice exhibited higher serum alanine aminotransferase levels compared with Xbp1(fl/fl) controls. RNA sequencing and Gene Ontogeny pathway analysis of hepatic mRNA revealed that apoptotic process, inflammatory response, and extracellular matrix structural constituent pathways had enhanced activation in HFS-fed Xbp1(-/-) mice. Liver histology demonstrated enhanced injury and fibrosis but less steatosis in the HFS-fed Xbp1(-/-) mice. Hepatic Col1a1 and Tgfβ1 gene expression, as well as Chop and phosphorylated JNK (p-JNK), were increased in Xbp1(-/-) compared with Xbp1(fl/fl) mice after HFS feeding. In vitro, stable XBP1-knockdown Huh7 cells (Huh7-KD) and scramble control cells (Huh7-SCR) were generated and treated with palmitic acid (PA) for 24 h. PA-treated Huh7-KD cells had increased cytotoxicity measured by lactate dehydrogenase release, apoptotic nuclei, and caspase3/7 activity assays compared with Huh7-SCR cells. CHOP and p-JNK expression was also increased in Huh7-KD cells following PA treatment. In conclusion, loss of XBP1 enhances injury in both in vivo and in vitro models of fatty liver injury. We speculate that hepatic XBP1 plays an important protective role in pathogenesis of NASH.
Project description:Nonalcoholic fatty liver disease is the most common chronic liver disease, which can progress to nonalcoholic steatohepatitis (NASH). Previous investigations demonstrated alterations in the expression and activity of hepatic drug transporters in NASH. Moreover, studies using rodent models of cholestasis suggest that compensatory changes in kidney transporter expression occur to facilitate renal excretion during states of hepatic stress; however, little information is currently known regarding extrahepatic regulation of drug transporters in NASH. The purpose of the current study was to investigate the possibility of renal drug transporter regulation in NASH across multiple experimental rodent models. Both rat and mouse NASH models were used in this investigation and include: the methionine and choline-deficient (MCD) diet, atherogenic diet, fa/fa rat, ob/ob and db/db mice. Histologic and pathologic evaluations confirmed that the MCD and atherogenic rats as well as the ob/ob and db/db mice all developed NASH. In contrast, the fa/fa rats did not develop NASH but did develop extensive renal injury compared with the other models. Renal mRNA and protein analyses of xenobiotic transporters suggest that compensatory changes occur in NASH to favor increased xenobiotic secretion. Specifically, both apical efflux and basolateral uptake transporters are induced, whereas apical uptake transporter expression is repressed. These results suggest that NASH may alter the expression and potentially function of renal drug transporters, thereby impacting drug elimination mechanisms in the kidney.
Project description:Background: Non-alcoholic fatty liver disease (NAFLD) is a common liver condition characterized by a significant accumulation of lipids in the liver without excessive alcohol consumption. Accumulating evidence suggests a significantly increased risk of intracerebral hemorrhage (ICH) in NAFLD patients. However, it remains poorly understood whether and how NAFLD affects the outcome of hemorrhagic brain injury. Here, we examined the effects of diet-induce NAFLD on ICH injury and neuroinflammation in mice. Methods: NAFLD was induced in C57BL/6 mice by feeding with a methionine-choline deficient (MCD) diet for 4 weeks. Collagenase and autologous blood models were used to evaluate the effects of NAFLD on ICH injury and neuroinflammation. Results: MCD diet for 4 weeks induces NAFLD and hyperlipidemia in mice. Mice receiving the MCD diet have aggravated neurological deficits and brain edema after ICH. The augmentation of ICH injury was accompanied by brain infiltration of neutrophils and monocytes and increased production of pro-inflammatory factors. Before ICH, MCD diet-induced mobilization of neutrophils and monocytes in the periphery. Notably, the detrimental effects of NAFLD on ICH injury was ablated in mice receiving antibody depletion of neutrophils and monocytes. Conclusions: These results suggest that NAFLD exacerbates neuroinflammation and ICH injury.
Project description:Testosterone signals through the androgen receptor (AR) and AR knockout mice develop obesity, suggesting a functional association between AR and leptin signaling. Furthermore, physiological blood concentrations of testosterone have been found to inhibit the development of arteriosclerosis, obesity and diabetes. However, these findings have not been verified by testosterone replacement in animal models and whether or not testosterone acts directly by activating AR to enhance leptin signaling, or indirectly by its conversion into estrogen remains unclear. Therefore, we investigated the effect of exogenously supplemented testosterone on glucose and lipid metabolism.Four-week-old male leptin receptor-knockout db/db mice were used as controls for a model of obesity retaining low testosterone. Mice were divided into sham-operated, castrated, or castrated and testosterone-supplemented groups and fed a high-fat diet (HFD) for 2 weeks from 5 weeks of age. Testosterone concentrations, blood glucose, plasma insulin levels, and intraperitoneal glucose tolerance and insulin tolerance were measured. At 7 weeks, triglyceride and glycogen content were measured in the liver and muscle. Lipid accumulation in the liver and soleus muscle was determined by immunohistochemistry with Oil Red O. Statistical analyses were performed using the Student's t-test or ANOVA where applicable.Lower testosterone levels in db/db mice compared with wild type (WT) db/+ mice were associated with glucose intolerance and fatty liver. Furthermore, castrated male db/db mice at 4 weeks of age progressively developed glucose intolerance accompanying a 15% increase in liver fat. Male mice fed a HFD had lower levels of testosterone compared with those fed a normal diet. We found that exogenous testosterone replacement injected subcutaneously into castrated male db/db mice alleviated the exacerbation of fatty liver and glucose intolerance, suggesting a leptin-independent mechanism. This mechanism is most likely mediated through gonadal axis suppression in this mouse model.In summary, testosterone may use a novel pathway to complement leptin signaling to regulate glucose and lipid metabolism, and thus offers a new therapeutic target to treat metabolic disorders.