Development Of Hepatic Steatosis Research Articles

Apoptosis-inducing factor deficient mice fail to develop hepatic steatosis under high fat high fructose diet or bile duct ligation.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein involved in redox signalling and programmed cell death. The role of AIF has been well recognized in diabetes and obesity. However, the aspect of AIF deficiency in the development of hepatic steatosis and liver injury is unknown. Therefore, in the current study, Harlequin (Hq mutant) mouse with markedly reduced content of AIF was investigated to explore the role of AIF on the initiation of liver injury. The wild type (WT) developed physiological and pathological features of non-alcoholic fatty liver disease (NAFLD) that were not seen in the Hq mice with AIF deficiency, when fed on high fat high fructose (HFHF) diet. Following bile duct ligation (BDL), the liver associated pathological changes were less conspicuous in Hq mice as compared to WT mice. The expression of AIF protein and apoptosis was markedly lesser as compared to their respective control in Hq mice on HFHF diet. Furthermore, the genes involved in fatty acid metabolism were also altered in the group of treated Hq mice. In conclusion, Hq mice failed to develop diet induced hepatic steatosis, suggestive of a role of AIF mediated pathway in the initiation and progression of liver inflammation. Thus, partial loss of AIF appears to be hepatoprotective. SIGNIFICANCE OF THE STUDY: AIF deficiency has multiple roles in altered pathology processes and cellular metabolism, thereby compromising the cellular homeostasis. Considering the molecular functions of AIF in other organ pathology little is known about its role in diet induced liver injury. Hence, the aim of the current study was to investigate the role of AIF deficiency in liver injury and diseases with focus on NAFLD. The study will help to deliniate the mechanisms of NAFLD using Harliquin Mice.

MiR-3666 inhibits development of hepatic steatosis by negatively regulating PPARγ

AimsPPARγ is a crucial transcription factor involved in development of hepatic steatosis, an early stage of NAFLD. PPARγ is tightly regulated through various positive and negative regulators including miRNAs. In this study, we report for the first time miR-3666 as a negative regulator of PPARγ and its involvement in development of hepatic steatosis. MethodsBinding of miR-3666 to regulate PPARγ was checked by luciferase assay and was confirmed by mutating PPARγ 3′UTR. Regulation of PPARγ was determined by overexpression of miR-3666 in HepG2 cells. Hepatic steatotic state in HepG2 cells was developed by exposure to excess palmitic acid and expression of PPARγ, miR-3666 and some PPARγ target and non-target genes was checked. Involvement of mir-3666 by regulating PPARγ in hepatic steatosis was also examined in liver of HFD fed mice. ResultsOn overexpression of miR-3666, PPARγ expression decreased significantly in a dose-dependent manner in HepG2 cells. Binding of miR-3666 to PPARγ was confirmed as the luciferase activity using pMIR-REPORT with PPARγ 3′UTR decreased in PA treated HepG2 cells overexpressing miR-3666 and remained unchanged when PPARγ 3′UTR was mutated. In PA treated HepG2 cells during development of hepatic steatosis PPARγ was significantly up-regulated concomitant with down-regulation of miR-3666. Overexpression of miR-3666 in these cells decreased the extent of hepatic steatosis. Significant up-regulation of PPARγ and down-regulation of miR-3666 was also observed in liver of HFD fed mice indicating that miR-3666 regulates PPARγ in vivo. ConclusionsmiR-3666 negatively regulates PPARγ by binding to its 3′UTR during development of hepatic steatosis.

Leptin decreases de novo lipogenesis in patients with lipodystrophy

De novo lipogenesis (DNL) plays a role in the development of hepatic steatosis. In humans with lipodystrophy, reduced adipose tissue causes lower plasma leptin, insulin resistance, dyslipidemia, and ectopic triglyceride (TG) accumulation. We hypothesized that recombinant leptin (metreleptin) for 6 months in 11 patients with lipodystrophy would reduce DNL by decreasing insulin resistance and glycemia, thus reducing circulating TG and hepatic TG. The percentage of TG in TG-rich lipoprotein particle (TRLP-TG) derived from DNL (%DNL) was measured by deuterium incorporation from body water into palmitate. At baseline, DNL was elevated, similar to levels previously shown in obesity-associated nonalcoholic fatty liver disease (NAFLD). After metreleptin, DNL decreased into the normal range. Similarly, absolute DNL (TRLP-TG × %DNL) decreased by 88% to near-normal levels. Metreleptin improved peripheral insulin sensitivity (hyperinsulinemic-euglycemic clamp) and lowered hemoglobin A1c and hepatic TG. Both before and after metreleptin, DNL positively correlated with insulin resistance, insulin doses, and hepatic TG, supporting the hypothesis that hyperinsulinemia stimulates DNL and that elevated DNL is integral to the pathogenesis of lipodystrophy-associated NAFLD. These data suggest that leptin-mediated improvement in insulin sensitivity increases clearance of blood glucose by peripheral tissues, reduces hepatic carbohydrate flux, and lowers insulinemia, resulting in DNL reductions and improvements in hepatic steatosis and dyslipidemia.

Eicosapentaenoic acid-containing polar lipids from seaweed Susabinori (Pyropia yezoensis) alleviate hepatic steatosis in obese db/db mice

Nonalcoholic fatty liver disease (NAFLD) is emerging as the most common liver disease in industrialized countries. Because hepatic steatosis is an early pathogenesis of NAFLD, the discovery of food components that could ameliorate hepatic steatosis is of interest. Susabinori (Pyropia yezoensis) is recognized as one of the most delicious edible brown algae, and we prepared lipid component of susabinori (SNL), which is rich in eicosapentaenoic acid (EPA)-containing polar lipids. In this study, we tested whether feeding SNL to db/db mice protects them from developing obesity-induced hepatic steatosis. After four weeks of feeding, hepatomegaly, hepatic steatosis, and hepatic injury were markedly alleviated in SNL-fed db/db mice. These effects were partly attributable to the suppression of activities and mRNA expressions of lipogenic enzymes and enhanced levels of adiponectin due to the SNL diet. Additionally, mRNA expression of monocyte chemoattractant protein-1, an inflammatory chemokine, was markedly suppressed, and the mRNA levels of PPARδ, the anti-inflammatory transcription factor, were strongly enhanced in the livers of db/db mice by the SNL diet. We speculate that the development and progression of obesity-induced hepatic steatosis was prevented by the suppression of chronic inflammation due to the combination of bioactivities of EPA, phospholipids, and glycolipids in the SNL diet.

Hepatic Steatosis After Neoadjuvant Chemotherapy for Pancreatic Cancer: Incidence and Implications for Outcomes After Pancreatoduodenectomy

BackgroundThis study aimed to determine the incidence of new onset hepatic steatosis after neoadjuvant chemotherapy for pancreatic cancer and its impact on outcomes after pancreatoduodenectomy. MethodsRetrospective review identified patients who received neoadjuvant chemotherapy for pancreatic adenocarcinoma and underwent pancreatoduodenectomy from 2013 to 2018. Preoperative computed tomography scans were evaluated for the development of hepatic steatosis after neoadjuvant chemotherapy. Hypoattenuation included liver attenuation greater than or equal to 10 Hounsfield units less than tissue density of spleen on noncontrast computed tomography and greater than or equal to 20 Hounsfield units less on contrast-enhanced computed tomography. ResultsOne hundred forty-nine patients received neoadjuvant chemotherapy for a median of 5 cycles (interquartile range (IQR), 4–6). FOLFIRINOX was the regimen in 78% of patients. Hepatic steatosis developed in 36 (24%) patients. The median time from neoadjuvant chemotherapy completion to pancreatoduodenectomy was 40 days (IQR, 29–51). Preoperative biliary stenting was performed in 126 (86%) patients. Neoadjuvant radiotherapy was delivered to 23 (15%) patients. Female gender, obesity, and prolonged exposure to chemotherapy were identified as risk factors for chemotherapy-associated hepatic steatosis. Compared with control patients without neoadjuvant chemotherapy-associated hepatic steatosis, patients developing steatosis had similar rates of postoperative pancreatic fistula (8% (control) vs. 4%, p = 0.3), delayed gastric emptying (8% vs. 14%, p = 0.4), and major morbidity (11% vs. 15%, p = 0.6). Ninety-day mortality was similar between groups (8% vs. 2%, p = 0.08). ConclusionHepatic steatosis developed in 24% of patients who received neoadjuvant chemotherapy but was not associated with increased morbidity or mortality after pancreatoduodenectomy.

Gut-liver axis modulation in fructose-fed mice: a role for PPAR-alpha and linagliptin.

Fructose dietary intake affects the composition of the intestinal microbiota and influences the development of hepatic steatosis. Endotoxins produced by gram-negative bacteria alter intestinal permeability and cause bacterial translocation. This study evaluated the effects of gut microbiota modulation by a purified PPAR-alpha agonist (WY14643), a DPP-4 inhibitor (linagliptin), or their association on intestinal barrier integrity, endotoxemia, and hepatic energy metabolism in high-fructose-fed C57BL/6 mice. Fifty mice were divided to receive the control diet (C group) or the high-fructose diet (HFRU) for 12 weeks. Subsequently, the HFRU group was divided to initiate the treatment with PPAR-alpha agonist (3.5 mg/kg/BM) and DPP-4 inhibitor (15 mg/kg/BM). The HFRU group had glucose intolerance, endotoxemia, and dysbiosis (with increased Proteobacteria) without changes in body mass in comparison with the C group. HFRU group showed damaged intestinal ultrastructure, which led to liver inflammation and marked hepatic steatosis in the HFRU group when compared to the C group. PPAR-alpha activation and DPP-4 inhibition countered glucose intolerance, endotoxemia, and dysbiosis, ameliorating the ultrastructure of the intestinal barrier and reducing Tlr4 expression in the liver of treated animals. These beneficial effects suppressed lipogenesis and mitigated hepatic steatosis. In conclusion, the results herein propose a role for PPAR-alpha activation, DPP-4 inhibition, and their association in attenuating hepatic steatosis by gut-liver axis modulation in high-fructose mice model. These observations suggest these treatments as potential targets to treat hepatic steatosis and avoid its progression.

Broad-spectrum antibiotics alter the microbiome, increase intestinal fxr, and decrease hepatic steatosis in zebrafish short bowel syndrome.

Short bowel syndrome (SBS) is associated with changes in the intestinal microbiome and marked local and systemic inflammation. There is also a late complication of SBS, intestinal failure associated liver disease (IFALD) in which hepatic steatosis progresses to cirrhosis. Most patients with SBS arrive at massive intestinal resection after a contaminating intraabdominal catastrophe and have a history of exposure to broad-spectrum antibiotics. We therefore investigated whether the administration of broad-spectrum antibiotics in conjunction with SBS in zebrafish (ZF) would replicate these systemic effects observed in humans to identify potentially druggable targets to aid in the management of SBS and resulting IFALD. In zebrafish with SBS, broad-spectrum antibiotics altered the microbiome, decreased inflammation, and reduced the development of hepatic steatosis. After two weeks of broad-spectrum antibiotics, these fish exhibited decreased alpha diversity, with less variation in microbial community composition between SBS and sham fish. Additionally, administration of broad-spectrum antibiotics was associated with decreased expression of intestinal toll-like receptor 4 (tlr4), increased expression of the intestinal gene encoding the Farnesoid X receptor (fxr), decreased expression of downstream hepatic cyp7a1, and decreased development of hepatic steatosis. SBS in zebrafish reproducibly results in increased epithelial surface area as occurs in human patients who demonstrate intestinal adaptation, but antibiotic administration in zebrafish with SBS reduced these gains with increased cell death in the intervillus pocket that contains stem/progenitor cells. These alternate states in SBS zebrafish might direct the development of future human therapies.NEW & NOTEWORTHY In a zebrafish model that replicates a common clinical scenario, systemic effects of the administration of broad-spectrum antibiotics in a zebrafish model of SBS identified two alternate states that led to the establishment of fat accumulation in the liver or its absence. Broad-spectrum antibiotics given to zebrafish with SBS over 2 wk altered the intestinal microbiome, decreased intestinal and hepatic inflammation, and decreased hepatic steatosis.

JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma

Metabolic stress causes activation of the cJun NH2-terminal kinase (JNK) signal transduction pathway. It is established that one consequence of JNK activation is the development of insulin resistance and hepatic steatosis through inhibition of the transcription factor PPARα. Indeed, JNK1/2 deficiency in hepatocytes protects against the development of steatosis, suggesting that JNK inhibition represents a possible treatment for this disease. However, the long-term consequences of JNK inhibition have not been evaluated. Here we demonstrate that hepatic JNK controls bile acid production. We found that hepatic JNK deficiency alters cholesterol metabolism and bile acid synthesis, conjugation, and transport, resulting in cholestasis, increased cholangiocyte proliferation, and intrahepatic cholangiocarcinoma. Gene ablation studies confirmed that PPARα mediated these effects of JNK in hepatocytes. This analysis highlights potential consequences of long-term use of JNK inhibitors for the treatment of metabolic syndrome.

Remnant Intestinal Length Defines Intestinal Adaptation and Hepatic Steatosis: Two Zebrafish Models

BackgroundShort bowel syndrome (SBS) is a condition that results from inadequate intestinal absorptive capacity, usually after the loss of functional intestine. We have previously developed a severe model of SBS in zebrafish that demonstrated increased intestinal adaptation (IA) and epithelial proliferation in SBS zebrafish. However, many children with SBS do not have this extreme intestinal loss. Therefore, in this study, we developed a variation of this model to evaluate the effects of increasing intestinal length on IA and the complications of SBS. Materials and methodsAfter Institutional Animal Care and Use Committee approval, adult male zebrafish were assigned to three groups: sham (n = 30), S1-SBS (n = 30), and S3-SBS (n = 30). Sham surgery included ventral laparotomy alone. S1-SBS surgery consisted of laparotomy with creation of a proximal stoma at S1 (jejunostomy equivalent) and ligation at S4. S3-SBS surgery had stoma creation at S3 (ileostomy equivalent) and the same ligation. Fish were harvested at 14 d. Markers of IA were measured from proximal intestinal segments, and the liver was analyzed for development of hepatic steatosis. ResultsAt 14 d, S3-SBS fish lost less weight than S1-SBS and had increased markers of IA compared with sham fish, which were decreased compared with S1-SBS fish. S3-SBS fish had decreased proximal intestinal inflammation compared with S1-SBS fish. S1-SBS fish developed extensive hepatic steatosis. Although S3-SBS fish have increased hepatic steatosis compared with sham fish, it is decreased compared with S1-SBS. ConclusionsLonger remnant intestine decreases the extent of IA, inflammation, and hepatic steatosis in a zebrafish model of SBS.

1849-P: Fructose Induces Hepatic Steatosis via ME1-Mediated Cytosolic Redox Status

Increased consumption of fructose-rich beverages has emerged as an important risk factor for development of hepatic steatosis, type 2 diabetes and metabolic syndrome. As is well-known, fructose robustly stimulates de novo lipogenesis in vivo; but how this occurs is less clarified and may involve multiple mechanisms. To determine the mechanism of fructose-induced hepatic steatosis and associated metabolic disease, we fed mice with chow diet and 30% fructose in drinking water. Fasting blood glucose level was elevated and insulin sensitivity was impaired in fructose fed group. Also, mice fed with fructose showed exaggerative lipid accumulation in the liver. To find out how fructose induced hepatic steatosis, we performed liver proteomic analysis following fructose fed. Compared with chow fed, Fructose fed significantly increased malic enzyme 1 (ME1), which is NADP-dependent and responsible for NADPH generation. Consistently, the ratio of NADPH/NADP+ in liver was increased by fructose fed, suggesting that fructose increased hepatic redox status. NADPH performed as a hydrogen donor for the synthesis of fatty acids, nucleic acids, etc. We also found that the multiple metabolites depending on NADPH production was dramatically enriched in our metabolome analysis. Then, we use selective ME1 inhibitor to decrease NAPDH production and redox status. The accumulation of triglyceride (TG) in primary hepatocytes by fructose was dose-dependently reduced by ME1 inhibitor. Similarly, the increasing of intracellular TG accumulation and NADPH/NADP+ ratio by fructose was inhibited upon ME1 knockdown by the application of siRNA. Taken together, our preliminary results suggested that ME1 mediated cytosolic redox status might play an important role in fructose induced hepatic steatosis and associated metabolic disease. Disclosure H. Jiang: None. J. Li: None.

Manipulation of Alcohol and Short-Chain Fatty Acids in the Metabolome of Commensal and Virulent Klebsiella pneumoniae by Linolenic Acid.

Endogenous alcohol produced by the gut microbiome is transported via the bloodstream to the liver for detoxification. Gut dysbiosis can result in chronic excess alcohol production that contributes to the development of hepatic steatosis. The aim of this study was to examine whether linolenic acid can manipulate the production of harmful alcohol and beneficial short-chain fatty acids (SCFAs) in the metabolome of commensal Klebsiella pneumoniae (K. pneumoniae) and the virulent K. pneumoniae K1 serotype. Glucose fermentation by the K. pneumoniae K1 serotype yielded increased production of alcohol and decreased SCFAs (especially acetate and propionate) compared to those of commensal K. pneumoniae. However, the use of linolenic acid instead of glucose significantly reduced alcohol and increased SCFAs in the fermentation media of the K. pneumoniae K1 serotype. The work highlights the value of shaping the microbial metabolome using linolenic acid, which can potentially regulate the gut–liver axis for the prevention and treatment of alcohol-induced liver diseases.

OR12-06 Nuclear Receptor CAR Protects Female Mice from the Development of Diet-Induced Nonalcoholic Fatty Liver Disease

NAFLD (Non Alcoholic Fatty Liver Disease) has become the most common cause of chronic liver disease in many developed countries worldwide and represents a major health concern. The prevalence of NAFLD is sexually dimorphic with men suspected to be more susceptible to the development of hepatic steatosis than women. Women are mostly protected until hormonal imbalance induced by menopause. Nuclear receptor CAR (Constitutive Androstan Receptor) is at the crossroads between endocrine and metabolic regulations and could therefore represent an interesting therapeutic target. It is primarily expressed in the liver and involved in the catabolism of hormones such as thyroid hormones, corticosteroids and estrogens. In addition, several studies reveal a metabolic role of CAR through regulation of major hepatic pathways such as neoglucogenesis, beta-oxidation and de novo lipogenesis. Our research is aimed at better understanding the role of CAR using a mouse model genetically deficient for CAR. To explore the metabolic functions of CAR, knock-out male and female mice were subjected to a high fat diet (HFD) for 16 weeks. Concomitant CAR deletion and high fat diet induces sexually dimorphic metabolic disorders. Knock-out of CAR in males exacerbates HFD-induced fasted hyperglycemia whereas in females, it aggravates body weight gain and adipose tissue accumulation. In accordance with epidemiological studies revealing a protection of women from the development of hepatic steatosis, HFD-fed WT female mice present less important hepatic steatosis than HFD-fed WT male mice. However, following CAR deletion, HFD-fed female mice develop a severe steatosis along with important hepatic injury. Ongoing studies aim to understand the transcriptomic and endocrine dysregulations that may explain these phenotypes. These results reveal a previously unrecognized dimorphic role of CAR in energy homeostasis and highlights its involvement in the protection of female mice towards the development of hepatic steatosis. Overall, this research provides further insights in the pathogenesis of NAFLD and its dimorphic prevalence.

OR17-01 Leptin Decreases De Novo Lipogenesis in Lipodystrophic Patients

De novo lipogenesis (DNL) plays a role in the development of hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). In rodent models of both health and lipodystrophy (LD), leptin decreases DNL. In human patients with LD, reduced adipose tissue results in adipokine deficiencies, including lower plasma leptin, which contributes to insulin resistance, dyslipidemia and ectopic accumulation of triglycerides (TG). The mechanisms by which leptin regulates serum and hepatic-TG are not well elucidated. Studying patients with LD before and after leptin therapy provides an important clinical model for understanding leptin’s effect on DNL. We hypothesized that leptin treatment in lipodystrophic patients would decrease DNL by decreasing insulin resistance and glycemia, resulting in reduced circulating and hepatic-TG.Leptin-naïve patients with LD (n=11) were treated with recombinant leptin (metreleptin) for 6 months. All measurements were performed after an 8–12 hr fast. The % of TG in TG-rich lipoproteins (TRLP-TG) derived from DNL (% DNL) was measured using body water labeling (oral D2O) of TG and mass spectrometry analysis. Absolute DNL was calculated as the product of TRLP-TG and % DNL. HbA1c and serum-TG were measured biochemically, hepatic-TG by MRI, and total body and hepatic insulin sensitivity measured during a hyperinsulinemic-euglycemic clamp.DNL decreased after metreleptin: % DNL from 22.8±6.8 to 9.1±5.1% (p=0.0008) and absolute DNL from 54.2±32.1 to 8.6±6.5 mg/dl (p=0.003). TRLP-TG decreased from (median [interquartile range]) 160 [107, 280] to 98 [66, 147] mg/dl (p=0.01). Total body and hepatic insulin sensitivity increased from 3.7 [3.0, 7.3] to 8.4 [5.1,10.6] mg/kgFFM/min (p=0.03) and from 61.0 [48.5, 69.3] to 84.7 [75.2, 107.6] % (p =0.01), respectively. HbA1c decreased from 8.6±1.8 to 7.1±1.4% (p=0.04), hepatic-TG decreased from 17.6±11.9 to 10.3±9.1% (p=0.02), and serum-TG from 386 [216, 686] to 223 [118, 497] mg/dl (p=0.06). DNL correlated negatively with insulin sensitivity both before (r=-0.73, p=0.03) and after (r=-0.85, p=0.004) metreleptin. DNL correlated positively with hepatic-TG before (r=0.70 p=0.03) and tended to correlate after metreleptin (r=0.65, p=0.06). The change in DNL correlated with change in serum-TG (r=0.77, p=0.04) but not the change in hepatic-TG (p=0.80).We show here for the first time that 6 months of metreleptin treatment in humans with LD decreased DNL by 84% and was associated with reductions in glycemia and improved peripheral and hepatic insulin sensitivity. These data indicate a strong link between metreleptin’s effects to increase clearance of blood glucose by peripheral tissues and reduce hepatic carbohydrate flux, resulting in DNL reductions. This led to lowered hepatic steatosis and dyslipidemia and suggests treatments that target multi-organ insulin resistance may lead to decreased NAFLD and cardiovascular risk.

Lack of Multidrug Resistance-associated Protein 4 Prolongs Partial Hepatectomy-induced Hepatic Steatosis.

Multidrug resistance-associated protein 4 (Mrp4) is an efflux transporter involved in the active transport of several endogenous and exogenous chemicals. Previously, we have shown that hepatic Mrp4 expression increases following acetaminophen overdose. In mice, these increases in Mrp4 expression are observed specifically in hepatocytes undergoing active proliferation. From this, we hypothesized that Mrp4 plays a key role in hepatocyte proliferation and that lack of Mrp4 impedes liver regeneration following liver injury and/or tissue loss. To evaluate the role of Mrp4 in these processes, we employed two-third partial hepatectomy (PH) as an experimental liver regeneration model. In this study, we performed PH-surgery on male wildtype (C57BL/6J) and Mrp4 knockout mice. Plasma and liver tissues were collected at 24, 48, and 72 h postsurgery and evaluated for liver injury and liver regeneration endpoints, and for PH-induced hepatic lipid accumulation. Our results show that lack of Mrp4 did not alter hepatocyte proliferation and liver injury following PH as evaluated by Ki-67 antigen staining and plasma alanine aminotransferase levels. To our surprise, Mrp4 knockout mice exhibited increased hepatic lipid content, in particular, di- and triglyceride levels. Gene expression analysis showed that lack of Mrp4 upregulated hepatic lipin1 and diacylglycerol O-acyltransferase 1 and 2 gene expression, which are involved in the synthesis of di- and triglycerides. Our observations indicate that lack of Mrp4 prolonged PH-induced hepatic steatosis in mice and suggest that Mrp4 may be a novel genetic factor in the development of hepatic steatosis.

Weight Regain in Formerly Obese Mice Hastens Development of Hepatic Steatosis Due to Impaired Adipose Tissue Function.

Weight regain after weight loss is common, and there is evidence to suggest negative effects on health because of weight cycling. This study sought to investigate the impact of weight regain in formerly obese mice on adipose tissue architecture and stromal cell function. A diet-switch model was employed for obesity induction, weight loss, and weight regain in mice. Flow cytometry quantified adipose tissue leukocytes in adipose tissue. Liver and adipose tissue depots were compared to determine tissue-specific effects of weight cycling. Epididymal white adipose tissue of formerly obese mice failed to expand in response to repeat exposure to high-fat diet and retained elevated numbers of macrophages and T cells. Weight regain was associated with disproportionally elevated liver mass, hepatic triglyceride content, serum insulin concentration, and serum transaminase concentration. These effects occurred despite an extended 6-month weightloss cycle and theydemonstrate that formerly obese mice maintain durable alterations in their physiological response to weight regain. Conditioned media from epididymal adipose tissue of formerly obese mice inhibited adipogenesis of 3T3-L1 preadipocytes, suggesting a potential mechanism to explain failed epididymal adipose tissue expansion during weight regain. Metabolic abnormalities related to defects in adipose tissue expansion and ongoing dysfunction manifest in formerly obese mice during weight regain.

Sex and BNIP3 genotype, rather than acute lipid injection, modulate hepatic mitochondrial function and steatosis risk in mice.

Both lipid oversupply and poor mitochondrial function (low respiration and elevated H2O2 emission) have been implicated in the development of hepatic steatosis and liver injury. Mitophagy, the targeted degradation of low-functioning mitochondria, is critical for maintaining mitochondrial quality control. Here, we used intralipid injection combined with acute (4 day) and chronic (4-7wk) high-fat diets (HFD) to examine whether hepatic mitochondrial respiration would decrease and H2O2 emission would increase with lipid overload. We tested these effects in male and female wild type (WT) mice and mice null for a critical mediator of mitophagy, BCL-2/adenovirus EIB 19-kDa interacting protein knockout (BNIP3 KO) housed at thermoneutral temperatures. Intralipid injection was successful in elevating serum triglycerides and nonesterified fatty acids but had no impact on hepatic mitochondrial respiratory function or H2O2 emission. However, female mice had greater mitochondrial respiration on the acute HFD and lower H2O2 emission across both HFD durations and were protected against hepatic steatosis. Unexpectedly, BNIP3 KO animals had greater hepatic mitochondrial respiration, better coupled respiration, and increased electron chain protein content after the 4-day HFD, compared with WT animals. Altogether, these data suggest that acute lipid overload delivered by a single intralipid bolus does not alter hepatic mitochondrial outcomes, but rather sex and genotype profoundly impact hepatic mitochondrial respiration and H2O2 emission.NEW & NOTEWORTHY This is the first study focusing on hepatic mitochondrial respiratory outcomes in response to lipid overload via a high-fat diet (HFD) combined with intralipid injection. Novel findings include no effect of intralipid injection on mitochondrial outcomes of interest despite increased circulating lipid concentrations. However, we report pronounced differences in hepatic mitochondrial respiration, complex protein expression, and H2O2 production by sex and BCL-2/adenovirus EIB 19-kDa interacting protein (BNIP3) genotype. Specifically, female mice had lower H2O2 emission globally and on an acute HFD, females had greater hepatic mitochondrial respiration than males while BNIP3 knockout (KO) animals had greater mitochondrial coupling and complex protein expression than wild-type (WT) animals.

PEG‐asparaginase‐induced hepatic steatosis is associated with PKA activation and white adipose tissue lipolysis

BackgroundPEGylated L‐asparaginase (PEG‐ASNase) is an essential chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia (ALL) that depletes L‐asparagine and leads to leukemic cell apoptosis. However, this agent is avoided in adult ALL protocols due to its high risk of liver toxicity. Clinical studies have implied that the mechanism of PEG‐ASNase‐induced liver toxicity is due to defect in hepatic β oxidation; however, no study has investigated the mechanism or the role of β oxidation in PEG‐ASNase‐induced hepatic toxicity. Our study aims to demonstrate that PEG‐ASNase‐induced liver toxicity is not due to hepatic defects in fatty acid oxidation, VLDL secretion, or fatty acid synthesis. Rather, our data suggest that the toxicity may be due to a novel mechanism of fatty liver involving drug‐induced adipose lipolysis.Methods8‐week‐old male Balb/c mice received 1,500 IU/kg of PEG‐ASNase and were sacrifice at 1, 3, 5, or 7 days after drug administration. Blood, liver, and white adipose tissue (WAT) were harvested after sacrifice. Liver lipids were directly quantified and further assessed via Oil red O and H&E staining. Total and direct bilirubin, ALT, AST, and non‐esterified fatty acids (NEFA) were measure in plasma. The mRNA and protein levels of genes involved in hepatic fatty acid oxidation, lipid synthesis, and VLDL secretion were determined. To assess the effect of PEG‐ASNase on WAT lipolysis, the protein levels of Atgl, phosphorylated Hsl, and protein kinase A (PKA) were measured.ResultsPEG‐ASNase treatment in mice led to total body, liver, and WAT weight loss. Mice developed hepatic microvesicular steatosis after 3–7 days of drug administration. Plasma direct and total bilirubin levels were elevated 8‐fold relative to controls, whereas ALT, AST, and NEFA concentrations were elevated 2‐fold 3–7 days after PEG‐ASNase. Furthermore, we found, that the hepatic mRNA and protein levels of Srebp‐1c and Fas, which are involved in fatty acid synthesis, were downregulated suggesting a decrease in lipid synthesis after PEG‐ASNase. Furthermore, the hepatic gene and protein expression of Pparα, Lcad, and Mcad, which are involved in fatty acid oxidation, were upregulated after PEG‐ASNase. In vivo VLDL secretion was also increased 3 days after PEG‐ASNase administration. Taken together, our data indicate that other lipid sources are mediating the drug‐induced fatty liver. Among WAT, we found that PEG‐ASNase elevated ATGL, phosphorylated HSL, and PKA protein levels, consistent with the WAT weight loss and the increased plasma NEFA levels.ConclusionOur data suggest that PEG‐ASNase‐induced WAT lipolysis leads to the development of microvesicular hepatic steatosis and toxicity. Our future studies will determine whether PEG‐ASNase hepatic steatosis can be inhibited via the pharmacological or genetic inhibition of adipose lipolysis and identify the molecular mechanism leading to PKA activation and lipolysis.Support or Funding InformationNIH Grants RO1 CA216815. University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261.

Intestinal gluconeogenesis prevents obesity-linked liver steatosis and non-alcoholic fatty liver disease

ObjectiveHepatic steatosis accompanying obesity is a major health concern, since it may initiate non-alcoholic fatty liver disease (NAFLD) and associated complications like cirrhosis or cancer. Intestinal gluconeogenesis (IGN) is a...

Ghrelin regulates adipose tissue metabolism: Role in hepatic steatosis

Fatty liver is the earliest and most common response of the liver to consumption of excessive alcohol. Steatosis can predispose the fatty liver to develop progressive liver damage. Chief among the many mechanisms involved in development of hepatic steatosis is dysregulation of insulin-mediated adipose tissue metabolism. Particularly, it is the enhanced adipose lipolysis-derived free fatty acids and their delivery to the liver that ultimately results in hepatic steatosis. The adipose-liver axis is modulated by hormones, particularly insulin and adiponectin. In recent studies, we demonstrated that an alcohol-induced increase in serum ghrelin levels impairs insulin secretion from pancreatic β-cells. The consequent reduction in circulating insulin levels promotes adipose lipolysis and mobilization of fatty acids to the liver to ultimately contribute to hepatic steatosis. Because many tissues, including adipose tissue, express ghrelin receptor we hypothesized that ghrelin may directly affect energy metabolism in adipocytes. We have exciting new preliminary data which shows that treatment of premature 3T3-L1 adipocytes with ghrelin impairs adipocyte differentiation and inhibits lipid accumulation in the tissue designed to store energy in the form of fat. We further observed that ghrelin treatment of differentiated adipocytes significantly inhibited secretion of adiponectin, a hepatoprotective hormone that reduces lipid synthesis and promotes lipid oxidation. These results were corroborated by our observations of a significant increase in serum adiponectin levels in ethanol-fed rats treated with a ghrelin receptor antagonist verses the un-treated ethanol-fed rats. Interestingly, in adipocytes, ghrelin also increases secretion of interleukin-6 (IL-6) and CCL2 (chemokine [C–C motif] ligand 2), cytokines which promote hepatic inflammation and progression of liver disease. To summarize, the alcohol-induced increase in serum ghrelin levels dysregulates adipose-liver interaction and promotes hepatic steatosis by increasing the free fatty acid released from adipose for hepatic uptake, and by altering adiponectin and cytokine secretion. Taken together, our data indicates that targeting the activity of ghrelin may be a powerful treatment strategy.

Interactions between resveratrol and gut microbiota affect the development of hepatic steatosis: A fecal microbiota transplantation study in high-fat diet mice

Abstract To investigate whether resveratrol could ameliorate the hepatic steatosis in high-fat diet (HFD) fed mice by modulating gut microbiota composition, fecal microbiota transplantation (FMT) was utilized in this study. Our results showed that resveratrol significantly ameliorated hepatic steatosis in HFD mice, increased the gene expression of fasting-induced adipose factor, and decreased the expression of lipogenesis-related genes and proteins (SREBP-1, FAS and ACC). In addition, resveratrol reversed HFD-induced gut microbiota dysbiosis, with an increase in the relative abundance of Bacteroidetes and a decrease in that of Firmicutes and Proteobacteria. Subsequent results showed the gut microbiota compositions of FMT recipient groups were similar to those of corresponding donor groups. And the changes of state of hepatic steatosis and expression of SREBP-1, ACC and FAS were coordinated with the results of gut microbiota. In conclusion, these results provided evidence to support the resveratrol could ameliorate the hepatic steatosis by modulating the gut microbiota.