Changes In Hepatic Expression Research Articles

Sucralose and stevia consumption leads to intergenerational alterations in body weight and intestinal expression of histone deacetylase 3

ObjectivesIt is unclear whether parental consumption of non-nutritive sweetener (NNS) can affect subsequent generations. The aim of this study was to determine whether chronic parental consumption of sucralose and stevia in mice affects body weight gain and liver and intestinal expression of histone deacetylase 3 (Hdac3) in these animals and in the subsequent first filial (F1) and second filial (F2) generations. MethodsMale and female mice (n = 47) were divided into three groups to receive water alone or supplemented with sucralose (0.1 mg/mL) or stevia (0.1 mg/mL) for 16 wk (parental [F0] generation). F0 mice were bred to produce the F1 generation; then, F1 mice were bred to produce the F2 generation. F1 and F2 animals did not receive NNSs. After euthanasia, hepatic and intestinal expression of Hdac3 was determined by quantitative reverse transcription polymerase chain reaction. ResultsBody weight gain did not differ between the three groups in the F0 generation, but it was greater in the F1 sucralose and stevia groups than in the control group. Consumption of both NNSs in the F0 generation was associated with lower Hdac3 expression in the liver and higher in the intestine. Hepatic Hdac3 expression was normalized to the control values in the F1 and F2 animals of the sucralose and stevia groups. Intestinal expression was still higher in the F1 generations of the sucralose and stevia groups but was partially normalized in the F2 generation of these groups, compared with control. ConclusionsNNS consumption differentially affects hepatic and intestinal Hdac3 expression. Changes in hepatic expression are not transmitted to the F1 and F2 generations whereas those in intestinal expression are enhanced in the F1 and attenuated in the F2 generations.

Protectin DX as a therapeutic strategy against frailty in mice

Frailty in aging is driven by the dysregulation of multiple biological pathways. Protectin DX (PDX) is a docosahexaenoic acid (DHA)-derived molecule that alleviates many chronic inflammatory disorders, but its potential effects on frailty remain unknown. Our goal is to identify age-related impairments in metabolic systems and to evaluate the therapeutic potential of PDX on frailty, physical performance, and health parameters. A set of 22-month-old C57BL/6 male and female mice were assigned to vehicle (Old) or PDX daily gavage treatment for 9 weeks, whereas 6-month-old (Adult) mice received only vehicle. Forelimb and hindlimb strength, endurance, voluntary wheel activity and walking speed determined physical performance and were combined with a frailty index score and body weight loss to determine frailty status. Our data shows that old vehicle-treated mice from both sexes had body weight loss paralleling visceromegaly, and Old females also had impaired insulin clearance as compared to the Adult group. Aging was associated with physical performance decline together with higher odds of frailty development. There was also age-driven mesangial expansion and glomerular hypertrophy as well as bone mineral density loss. All of the in vivo and in vitro impairments observed with aging co-occurred with upregulation of inflammatory pathways and Myc signaling as well as downregulation of genes related to adipogenesis and oxidative phosphorylation in liver. PDX attenuated the age-driven physical performance (strength, exhaustion, walking speed) decline, promoted robustness, prevented bone losses and partially reversed changes in hepatic expression of Myc targets and metabolic genes. In conclusion, our data provides evidence of the beneficial therapeutic effect of PDX against features of frailty in mice. Further studies are warranted to investigate the mechanisms of action and the potential for human translation.

Differential effects of continuous and intermittent daytime food deprivation periods on metabolism and reproductive performance in diurnal zebra finches

We investigated whether food availability effects on metabolism and reproduction are the result of the sum effect of daily feeding (food availability) and starvation (food deprivation) periods. Adult zebra finches were paired and subjected to a time-restricted feeding (TRF) regimen consisting of continuous and intermittent daytime food deprivation periods. Birds were given food during the 12-h day for a total of 4-h in the evening (1 *4-h, hour 8–12), or in 2 splits of 2 h each (2 * 2-h) or 4 splits 1 h each (4 * 1-h), with controls on food ad libitum, until they had the first egg clutch. TRF caused significant changes in hepatic expression of metabolism-associated sirt1, egr1, pparα and foxo1 genes despite no difference in the food intake, body mass and blood glucose levels. Importantly, TRF resulted in a significant reduction in plasma testosterone and estradiol levels, delayed nest-building and egg laying, and reduced clutch size. Concurrently, under TRF regimes, we found a significantly lower expression of th and mtr genes linked with motivation and affiliation (but not of dio2, dio3, gnrh1 and gnih genes linked with gonadal maturation) in the hypothalamus, and of star and hook 1 genes in the testes and star, cyp19 and erα genes in the ovary. These results demonstrate the importance of daily food deprivation times on the metabolism and reproduction, and suggest a possible provisioning of energy available from daily feeding for the maintenance of body condition at the expense of reproduction performance in diurnal animals.

Daytime restriction of feeding prevents illuminated night-induced impairment of metabolism and sleep in diurnal zebra finches

We investigated whether nocturnal eating was causal to the impairment of metabolism and sleep disruption in diurnal animals exposed to illuminated nights. Adult zebra finches hatched and raised in 12 h light: 12 h darkness (LD) were exposed to 5-lux dim light at night (dLAN, two groups), with a control group maintained on LD. For the next 3 weeks, the food availability to one of the dLAN groups was restricted to the 12 h light period (dLAN -F); the other dLAN (dLAN +F) and LD groups were continued on ad lib feeding. In spite of similar food intakes, dLAN +F condition led to the fat accumulation and weight gain. These birds showed concurrent changes in hepatic expression of genes associated with carbohydrate and lipid metabolism, suggesting an enhanced gluconeogenesis and impaired fatty acids synthesis. Increased sirt1 mRNA levels indicated the activation of molecular mechanisms to counter-balance the metabolic damage under dLAN +F. Furthermore, reduced bout length and total duration of the nocturnal sleep suggested a poorer sleep in dLAN +F condition. Negative sleep effects of dLAN were supported by the lower hypothalamic expression of sleep promoting sik3 and camkii genes, and higher mRNA expression of awake promoting achm3 gene in dLAN +F, compared to the LD condition. Importantly, dLAN-induced negative effects in metabolism and sleep were alleviated in the dLAN –F group. These results suggest the role of timed feeding in alleviating the negative impact of illuminated nights in metabolism and sleep in diurnal zebra finches.

Squalene through Its Post-Squalene Metabolites Is a Modulator of Hepatic Transcriptome in Rabbits.

Squalene is a natural bioactive triterpene and an important intermediate in the biosynthesis of sterols. To assess the effect of this compound on the hepatic transcriptome, RNA-sequencing was carried out in two groups of male New Zealand rabbits fed either a diet enriched with 1% sunflower oil or the same diet with 0.5% squalene for 4 weeks. Hepatic lipids, lipid droplet area, squalene, and sterols were also monitored. The Squalene administration downregulated 9 transcripts and upregulated 13 transcripts. The gene ontology of transcripts fitted into the following main categories: transporter of proteins and sterols, lipid metabolism, lipogenesis, anti-inflammatory and anti-cancer properties. When the results were confirmed by RT-qPCR, rabbits receiving squalene displayed significant hepatic expression changes of LOC100344884 (PNPLA3), GCK, TFCP2L1, ASCL1, ACSS2, OST4, FAM91A1, MYH6, LRRC39, LOC108176846, GLT1D1 and TREH. A squalene-enriched diet increased hepatic levels of squalene, lanosterol, dihydrolanosterol, lathosterol, zymostenol and desmosterol. Strong correlations were found among specific sterols and some squalene-changed transcripts. Incubation of the murine AML12 hepatic cell line in the presence of lanosterol, dihydrolanosterol, zymostenol and desmosterol reproduced the observed changes in the expressions of Acss2, Fam91a1 and Pnpla3. In conclusion, these findings indicate that the squalene and post-squalene metabolites play important roles in hepatic transcriptional changes required to protect the liver against malfunction.

β2-Adrenergic receptor agonist induced hepatic steatosis in mice: modeling nonalcoholic fatty liver disease in hyperadrenergic states.

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from hepatic steatosis [excessive accumulation of triglycerides (TG)] to nonalcoholic steatohepatitis, which can progress to cirrhosis and hepatocellular carcinoma. The molecular pathogenesis of steatosis and progression to more severe NAFLD remains unclear. Obesity and aging, two principal risk factors for NAFLD, are associated with a hyperadrenergic state. β-Adrenergic responsiveness in liver increases in animal models of obesity and aging, and in both is linked to increased hepatic expression of β2-adrenergic receptors (β2-ARs). We previously showed that in aging rodents intracellular signaling from elevated hepatic levels of β2-ARs may contribute to liver steatosis. In this study we demonstrate that injection of formoterol, a highly selective β2-AR agonist, to mice acutely results in hepatic TG accumulation. Further, we have sought to define the intrahepatic mechanisms underlying β2-AR mediated steatosis by investigating changes in hepatic expression and cellular localization of enzymes, transcription factors, and coactivators involved in processes of lipid accrual and disposition-and also functional aspects thereof-in livers of formoterol-treated animals. Our results suggest that β2-AR activation by formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, increased but incomplete β-oxidation of fatty acids with accumulation of potentially toxic long-chain acylcarnitine intermediates, and reduced TG secretion-all previously invoked as contributors to fatty liver disease. Experiments are ongoing to determine whether sustained activation of hepatic β2-AR signaling by formoterol might be utilized to model fatty liver changes occurring in hyperadrenergic states of obesity and aging, and thereby identify novel molecular targets for the prevention or treatment of NAFLD.NEW & NOTEWORTHY Results of our study suggest that β2-adrenergic receptor (β2-AR) activation by agonist formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, incomplete β-oxidation of fatty acids with accumulation of long-chain acylcarnitine intermediates, and reduced TG secretion. These findings may, for the first time, implicate a role for β2-AR responsive dysregulation of hepatic lipid metabolism in the pathogenetic processes underlying NAFLD in hyperadrenergic states such as obesity and aging.

Depletion and enrichment of phytosterols in soybean oil lipid emulsions directly associate with serum markers of cholestasis in preterm parenteral nutrition-fed pigs.

Clinical reports show a positive correlation between phytosterol concentrations and severity of cholestatic liver disease markers in infants during long-term administration of parenteral lipid emulsions. Establishing a causal link between phytosterols and cholestasis has been complicated by confounding factors of lipid emulsion load, fatty acid composition, and vitamin E in many of these studies. The goal of this study is to determine whether altering the phytosterol concentration within a common soybean oil-based emulsion will alter the onset and severity of cholestasis in parenterally fed preterm piglets. Preterm piglets were administered, for 21 days, either enteral nutrition (ENT) or parenteral nutrition (PN) prepared from a soybean oil-based emulsion containing either 24.0% (depleted [DEP]), 100% (Intralipid; normal phytosterol [NP] concentration), or 144% (enriched [ENR]) total phytosterol concentration. At the end of the study, plasma and liver phytosterol concentrations were highest in the ENR group, followed by NP and then DEP and ENT. Serum direct bilirubin, serum bile acids, and γ-glutamyltransferase were higher in the ENR and NP groups compared with either DEP or ENT groups. All PN lipid groups showed evidence of mild hepatic steatosis but no change in hepatic expression of proinflammatory cytokines or Farnesoid X receptor target genes. The increase in serum direct bilirubin was lower in the DEP group vs the lipid emulsions with normal or ENR phytosterols. Our results provide additional evidence that phytosterols are linked to an increase in serum markers of cholestasis in preterm PN-fed pigs.

Effects of tesamorelin on hepatic transcriptomic signatures in HIV-associated NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is a common comorbidity among people living with HIV that has a more aggressive course than NAFLD among the general population. In a recent randomized placebo-controlled trial, we demonstrated that the growth hormone–releasing hormone analog tesamorelin reduced liver fat and prevented fibrosis progression in HIV-associated NAFLD over 1 year. As such, tesamorelin is the first strategy that has shown to be effective against NAFLD among the population with HIV. The current study leveraged paired liver biopsy specimens from this trial to identify hepatic gene pathways that are differentially modulated by tesamorelin versus placebo. Using gene set enrichment analysis, we found that tesamorelin increased hepatic expression of hallmark gene sets involved in oxidative phosphorylation and decreased hepatic expression of gene sets contributing to inflammation, tissue repair, and cell division. Tesamorelin also reciprocally up- and downregulated curated gene sets associated with favorable and poor hepatocellular carcinoma prognosis, respectively. Notably, among tesamorelin-treated participants, these changes in hepatic expression correlated with improved fibrosis-related gene score. Our findings inform our knowledge of the biology of pulsatile growth hormone action and provide a mechanistic basis for the observed clinical effects of tesamorelin on the liver.

Evidence of increased hypoxia signaling in fetal liver from maternal nutrient restriction in mice.

Intrauterine growth restriction (IUGR) is a pregnancy condition where fetal growth is reduced, and offspring from IUGR pregnancies are at increased risk for type II diabetes as adults. The liver is susceptible to fetal undernutrition experienced by IUGR infants and animal models of growth restriction. This study aimed to examine hepatic expression changes in a maternal nutrient restriction (MNR) mouse model of IUGR to understand fetal adaptations that influence adult metabolism. Liver samples of male offspring from MNR (70% of ad libitum starting at E6.5) or control pregnancies were obtained at E18.5 and differential expression was assessed by RNAseq and western blots. Forty-nine differentially expressed (FDR < 0.1) transcripts were enriched in hypoxia-inducible pathways including Fkbp5 (1.6-fold change), Ccng2 (1.5-fold change), Pfkfb3 (1.5-fold change), Kdm3a (1.2-fold change), Btg2 (1.6-fold change), Vhl (1.3-fold change), and Hif-3a (1.3-fold change) (FDR < 0.1). Fkbp5, Pfkfb3, Kdm3a, and Hif-3a were confirmed by qPCR, but only HIF-2a (2.2-fold change, p = 0.002) and HIF-3a (1.3 p = 0.03) protein were significantly increased. Although a moderate impact, these data support evidence of fetal adaptation to reduced nutrients by increased hypoxia signaling in the liver.

Protein Kinase C Epsilon Deletion in Adipose Tissue, but Not in Liver, Improves Glucose Tolerance

Protein kinase C epsilon (PKCɛ) activation in the liver is proposed to inhibit insulin action through phosphorylation of the insulin receptor. Here, however, we demonstrated that global, but not liver-specific, deletion of PKCɛ in mice protected against diet-induced glucose intolerance and insulin resistance. Furthermore, PKCɛ-dependent alterations in insulin receptor phosphorylation were not detected. Adipose-tissue-specific knockout mice did exhibit improved glucose tolerance, but phosphoproteomics revealed no PKCɛ-dependent effect on the activation of insulin signaling pathways. Altered phosphorylation of adipocyte proteins associated with cell junctions and endosomes was associated with changes in hepatic expression of several genes linked to glucose homeostasis and lipid metabolism. The primary effect of PKCɛ on glucose homeostasis is, therefore, not exerted directly in the liver as currently posited, and PKCɛ activation in this tissue should be interpreted with caution. However, PKCɛ activity in adipose tissue modulates glucose tolerance and is involved in crosstalk with the liver.

Age-related changes in hepatic expression and activity of drug metabolizing enzymes in male wild-type and breast cancer resistance protein knockout mice.

This study aimed to reveal age-related changes in the expression and activity of seven hepatic drug metabolizing enzymes (DMEs) in male wild-type and breast cancer resistance protein knockout (Bcrp1-/- ) FVB mice. The protein expression of four cytochrome P450 (Cyps) (Cyp3a11, 2d22, 2e1, and 1a2), and three UDP-glucuronosyltransferases (Ugts) (Ugt1a1, 1a6a, and 1a9) in liver microsomes of wild-type and Bcrp1-/- FVB mice at different ages were determined using a validated ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method. The activities and mRNA levels of these DMEs were measured using the probe substrates method and real-time PCR, respectively. In the liver of wild-type FVB mice, Cyp3a11, 2d22, 2e1, 1a2, Ugt1a1, and 1a6a displayed maximum protein levels at 6-9weeks of age. Cyp1a2, Ugt1a1, 1a6a, and 1a9 showed maximum activities at 6-9weeks of age, whereas Cyp3a11, 2d22, and 2e1 showed maximum activities in 1-3-week-old mice. Additionally, most of the DMEs showed maximum mRNA levels in 17-week-old mice liver. Compared with wild-type FVB mice, the protein levels of these DMEs showed no significant changes in Bcrp1-/- FVB mice liver. However, the activity of Cyp2e1 was increased and that of Cyp2d22 was decreased. In conclusion, the seven hepatic DMEs in FVB mice liver showed significant alterations in an isoform-specific manner with increased age. Although the protein levels of these DMEs showed no significant changes, the activities of Cyp2e1 and 2d22 were changed in Bcrp1-/- mice.

Changes in Hepatic Expression of Hyaluronan Synthases (HAS2 and HAS3) After Treatment with Steptozotocin in Channel Catfish

Streptozotocin (STZ) is commonly used experimentally to induce hyperglycemia in rodents. As an alternative model organism for investigating human obesity, channel catfish show a genetic predisposition toward an accelerated growth rate resulting in a phenotype similar to obese humans. In our previous studies, STZ treatment resulted in hypoglycemia and abnormal liver morphology in channel catfish, indicating that STZ treatment possibly induces liver damage. The mechanisms involved in the STZ‐induced hypoglycemic and tissue damage have never been examined. Another noted marker for liver damage stems from hyaluronan synthases (HAS), which have been associated with tissue damage in both humans and rodents. We hypothesized the expression of HAS mRNA would change in response to liver damage induced by STZ in channel catfish. Juvenile channel catfish were treated with 0, 3.6, 36, 180, or 360 mg STZ/kg body weight (n=4 fish/treatment) on day 0. Liver samples were collected on day 7. Hepatic expression of HAS2 and HAS3 mRNA was examined, using real‐time PCR. Expression of HAS2 and HAS3 mRNA decreased in response to an increased dose of STZ (p &lt; 0.05 and p &lt; 0.10, respectively). These results suggest STZ induces severe liver damage that down regulates the expression of HAS2 and HAS3 mRNA. However, the effects of STZ on gene expression appear to be global, and the tissue damage induced by STZ is not reversible by changes in the amount or activity of HAS.Support or Funding InformationThis research was funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS) (Grant #IOS‐1238831). It was also supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nutritional depletion in n-3 PUFA in apoE knock-out mice: A new model of endothelial dysfunction associated with fatty liver disease.

Western diets are characterized by low intake of n-3 PUFA compensated by constant amounts of n-6 PUFA. Reduced intake of n-3 PUFA is associated with increased cardiovascular risk, as observed in nonalcoholic fatty liver disease patients. The study aimed to evaluating the impact of dietary n-3 PUFA depletion on endothelial function, an early key event of cardiovascular diseases. C57Bl/6J or apolipoprotein E knock-out (apoE-/- ) were fed control (CT) or n-3 PUFA-depleted diets (DEF) for 12 wks. Mice fed n-3 DEF diet developed a hepatic steatosis, linked to changes in hepatic expression of genes controlled by Sterol Regulatory Element Binding Protein-1 and -2. Vascular function was assessed on second- and third-order mesenteric arteries and n-3 PUFA-depleted apoE-/- mice presented endothelial dysfunction characterized by decreased vasorelaxation in response of acetylcholine. The presence of a nitric oxide synthase (NOS) inhibitor blunted the relaxation in each groups and heme-nitrosylated hemoglobin blood (Hb-NO) level was significantly lower in n-3 PUFA-depleted apoE-/- mice. Twelve weeks of n-3 DEF diet promote steatosis and accelerate the process of endothelial dysfunction in apoE-/- mice by a mechanism involving the NOS/NO pathway. We propose n-3 PUFA-depleted apoE-/- mice as a new model to study endothelial dysfunction related to hepatic steatosis independently of obesity.

Abstract 24: Tubular Vegfa is Required for Renal Microvasculature and Oxygen Sensing

Adequate renal oxygenation is pivotal for maintaining essential functions of the kidney. Vascular endothelial growth factor A (VEGFA) plays a key role in vascular formation and maintenance, processes that are critical for sufficient tissue perfusion and oxygenation. Here we show that Vegfa is expressed in select tubular epithelial cells within the nephron, while expression of its receptor ( Kdr/Vegfr2 ) is restricted largely to capillary beds. Early genetic ablation of tubular Vegfa allows the formation of a grossly normal kidney. However, microvascular density is markedly reduced, leading to increased renal hypoxia. As a consequence the mice develop pronounced polycythemia (81 ± 1.62 vs. 58 ± 0.87 % Hematocrit), due to an increase in serum erythropoietin concentration (426.5 ± 49.3 vs. 249.3 ± 13.6 pg/ml). The elevation in circulating erythropoietin is elicited by an augmented renal production of the hormone, and occurs independent of changes in hepatic expression. In kidney, expression of classical hypoxia-inducible factor-1 responsive transcripts remains unaltered; suggesting that erythropoiesis partly compensates the capillary rarefaction. An increase in diastolic blood pressure (DBP, 112 ± 3.7 vs. 104 ± 1.6 mmHg) is present in mice lacking tubular Vegfa maintained on a normal diet, while systolic blood pressure (SBP, 139 ± 2.8 vs. 135 ± 2.8 mmHg) does not change. The difference in diastolic pressure is ablated when dietary NaCl content changed (DBP: 107 ± 3.1 vs. 103 ± 2.7 mmHg; SBP: 132 ± 4.1 vs. 131 ± 2.9 mmHg; for low NaCl), or when angiotensin II is infused (DBP: 137 ± 6.7 vs. 134 ± 3.8 mmHg; SBP: 161 ± 7.6 vs. 162 ± 9.3 mmHg). Moreover, renal electrolyte excretion remains unperturbed by reductions in tubular Vegfa. In conclusion, tubular Vegfa is critical for adequate development of renal microvasuculature, a prerequisite for proper oxygenation of the kidney. Compensatory polycythemia likely prevents severe perturbations of renal function in this model. In addition, increased diastolic blood pressure may occur as a consequence of capillary rarefaction within the kidney.

Maternal Exercise During Pregnancy Improves Glucose Metabolism of Offspring

The intrauterine environment during pregnancy is a critical factor in the development of diabetes and obesity in offspring. To determine the effects of maternal exercise during pregnancy on the metabolic health of offspring, 6wk old C57BL/6 virgin female mice were fed a chow or high‐fat (HF) (60%) diet and divided into 4 sub‐groups: Trained (housed with running wheels for 2wks preconception and during gestation); Pre‐Pregnancy Trained (housed with running wheels for 2wks pre‐conception); Gestation Trained (housed with running wheels during gestation); or Sedentary (static cages). Breeders were sedentary and chow‐fed. Offspring were sedentary, chow‐fed and studied for 12 months. From 6 months on, male offspring from Trained dams had decreased serum insulin concentrations, improved glucose tolerance, and decreased % body fat vs. all other groups. This improved metabolic profile occurred in offspring of both chow‐ and HF‐fed dams. Female offspring had similar responses. 12 month old offspring from Trained dams had significant changes in hepatic expression of key genes regulating Krebs cycle activity and fatty acid oxidation, suggesting liver adaptations are central for this effect. In conclusion, maternal exercise before and during pregnancy in mice has profound effects to improve the metabolic health of offspring. Thus, maternal exercise may combat the increasing rates of diabetes and obesity.

Induction of apolipoprotein A-I gene expression by glucagon-like peptide-1 and exendin-4 in hepatocytes but not intestinal cells

ObjectiveDiabetic dyslipidemia is an important risk factor for the development of macrovascular complications. Recent clinical trials suggest that diabetics treated with glucagon-like peptide-1 (GLP-1) have normalized lipid levels, including an increase in plasma high-density lipoprotein cholesterol (HDLc) levels. MethodsTo determine if GLP-1 (7-36 amide) and the GLP-1-like insulinotropic peptide exendin-4 regulate expression of apolipoprotein A-I (apo A-I), the primary anti-atherogenic component of high-density lipoprotein (HDL), HepG2 hepatocytes and Caco-2 intestinal cells, representative of tissues that express the majority of apo A-I, were treated with increasing amounts of each peptide and apo A-I gene expression was measured in the conditioned medium. ResultsApo A-I secretion increased in both GLP-1 and exendin-4-treated HepG2, but not Caco-2 cells, and this was accompanied by similar changes in apo A-I mRNA levels and apo A-I promoter activity. Induction of apo A-I promoter activity by GLP-1 and exendin-4 required an SP1-responsive element. Hepatic ATP binding cassette protein A1 (ABCA1) expression, but not scavenger receptor class B type1 receptor expression was also induced by GLP-1 and exendin-4. ConclusionsThese results suggest that GLP-1- and exendin-4-mediated changes in HDLc are likely due to changes in hepatic expression of apo A-I and ABCA1.

IRS‐1/2 Mediated Regulation of Hepatic OGT and OGA Expression

To investigate the role of hepatic insulin signaling in the postprandial regulation of O‐GlcNAc transferase (OGT) and OGlcNAcase (OGA) expression, C57BL/6 male mice with a conditional knock out of IRS‐1/2 in liver (LKO) were fed a chow or high fat diet (HFD) for 16 weeks and then either fasted for 18hrs or fasted and refed ad libitum for 4hrs. In control animals that were fasted or refed, qRT‐PCR revealed a postprandial increase in hepatic OGT expression (146% ± 12.8, n=5) and a reduction in OGA transcript levels (65.7% ± 6.1, n=5). The postprandial increase in hepatic OGT expression was attenuated in LKO mice. In control mice, high fat feeding attenuated both the postprandial rise in hepatic OGT expression (123% ± 5.0, n=5) and the suppression of OGA expression (77.2% ± 4.6, n=5). In LKO mice in the refed state, OGT mRNA expression was reduced by 68% ± 1.5 (n=5) after 16 weeks HFD as compared to animals fed a chow diet. Furthermore with 16 weeks HFD, elevated expression of OGA was observed in hepatic (170% ± 7.5) and adipose (316.5% ± 21.6, n=5) tissue as compared to chow diet. These studies reveal that the postprandial changes in hepatic expression of OGA and OGT are regulated, in part, by IRS‐1/2 mediated insulin signaling in vivo and that the suppression of OGA expression in fat fed animals is lost in the absence of hepatic IRS‐1/2. Supported by T32 HL07260 and RO1DK002001.

Toxicogenomic analysis of exposure to TCDD, PCB126 and PCB153: identification of genomic biomarkers of exposure to AhR ligands

BackgroundTwo year cancer bioassays conducted by the National Toxicology Program have shown chronic exposure to dioxin-like compounds (DLCs) to lead to the development of both neoplastic and non-neoplastic lesions in the hepatic tissue of female Sprague Dawley rats. Most, if not all, of the hepatotoxic effects induced by DLC's are believed to involve the binding and activation of the transcription factor, the aryl hydrocarbon receptor (AhR). Toxicogenomics was implemented to identify genomic responses that may be contributing to the development of hepatotoxicity in rats.ResultsThrough comparative analysis of time-course microarray data, unique hepatic gene expression signatures were identified for the DLCs, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (100 ng/kg/day) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) (1000 ng/kg/day) and the non-DLC 2,2',4,4',5,5',-hexachlorobiphenyl (PCB153) (1000 μg/kg/day). A common time independent signature of 41 AhR genomic biomarkers was identified which exhibited at least a 2-fold change in expression following subchronic (13-wk) and chronic (52-wk) p.o. exposure to TCDD and PCB126, but not the non DLC, PCB153. Real time qPCR analysis validated that 30 of these genes also exhibited at least a 2-fold change in hepatic expression at 24 hr following a single exposure to TCDD (5 μg/kg, po). Phenotypic anchoring was conducted which identified forty-six genes that were differently expressed both following chronic p.o. exposure to DLCs and in previously reported studies of cholangiocarcinoma or hepatocellular adenoma.ConclusionsTogether these analyses provide a comprehensive description of the genomic responses which occur in rat hepatic tissue with exposure to AhR ligands and will help to isolate those genomic responses which are contributing to the hepatotoxicity observed with exposure to DLCs. In addition, the time independent gene expression signature of the AhR ligands may assist in identifying other agents with the potential to elicit dioxin-like hepatotoxic responses.

Pioglitazone improves lipid and insulin levels in overweight rats on a high cholesterol and fructose diet by decreasing hepatic inflammation.

Nutrient overload leads to obesity and insulin resistance. Pioglitazone, a selective peroxisome proliferator-activated receptor (PPAR)gamma agonist, is currently used to manage insulin resistance, but the specific molecular mechanisms activated by PPARgamma are not yet fully understood. Recent studies suggest the involvement of suppressor of cytokine signalling (SOCS)-3 in the pathogenesis of insulin resistance. This study aimed to investigate the hepatic signalling pathway activated by PPARgamma activation in a non-genetic insulin-resistant animal model. Male Wistar rats were maintained on a high-cholesterol fructose (HCF) diet for 15 weeks. Pioglitazone (3 mg x kg(-1)) was administered orally for the last 4 weeks of this diet. At the end of the treatment, serum was collected for biochemical analysis. Levels of PPARgamma, SOCS-3, pro-inflammatory markers, insulin receptor substrate-2 and Akt/glycogen synthase kinase-3beta phosphorylation were assessed in rat liver. Rats fed the HCF diet exhibited hyperlipidemia, hyperinsulinemia, impaired glucose tolerance and insulin resistance. Pioglitazone administration evoked a significant improvement in lipid metabolism and insulin responsiveness. This was accompanied by reduced hepatic expression of SOCS-3, interleukin-6, tumour necrosis factor-alpha and markers of neutrophil infiltration. Diet-induced PPARgamma expression was unaffected by the pioglitazone treatment. Chronic pioglitazone administration reduced hepatic inflammatory responses in rats fed a HCF diet. These effects were associated with changes in hepatic expression of SOCS-3, which may be a crucial link between the reduced local inflammation and the improved insulin signalling.

Inhibition of inducible nitric oxide synthase worsens liver damage regardless of lipopolysaccharide treatment in small-for-size liver transplantation

Objective In small-for-size liver transplantation, portal hypertension aggravates endotoxin from the gut which accelerates the activation of inducible nitric oxide synthase (iNOS). However, there is little knowledge as to the effects of iNOS inhibitors on small-for-size graft damage. Our study was designed to investigate the role of an iNOS inhibitor both with and without lipopolysaccharide (LPS) treatment in ischemia–reperfusion injury of small-for-size liver transplantation. Methods Subjecting Sprague–Dawley rats to small-for-size grafts liver transplantation, we investigated the time course of changes in hepatic expression of iNOS and endothelial nitric oxide synthase (eNOS). Meantime, we also investigated the effects of iNOS inhibitor, both with and without LPS treatment, at 6 h after reperfusion. Results While iNOS mRNA expression reached a peak at 3 h, the highest protein level occurred at 6 h after reperfusion. Aminoguanidine (AG) significantly inhibited mRNA and protein expressions of iNOS, but not that of eNOS. However, LPS accelerated activation of iNOS, but suppressed the expression of eNOS. Meanwhile, compared with the untreated group, those treated with AG or LPS experienced worsened liver function and tissue damage, promoting neutrophil infiltration in the liver tissue. The difference between the LPS group and the LPS + AG group was found to be significant. In addition, AG and LPS treatments up-regulated the protein expression of ICAM-1 and NF-κB p65. Conclusion In a small-for-size model of rat liver transplantation, regardless of LPS treatment, the inhibitor of iNOS, AG, attenuated iNOS expression, but worsened liver function and tissue damage. The subsequent increased neutrophil infiltration in liver tissue may be associated with up-regulation of ICAM-1 and NF-κB expressions.