Increased Triglyceride Accumulation Research Articles

Chronic Exposure to Rifaximin Causes Hepatic Steatosis in Pregnane X Receptor-Humanized Mice

Rifaximin, a nonsystemic antibiotic that exhibits low gastrointestinal absorption, is a potent agonist of human pregnane X receptor (PXR), which contributes to its therapeutic efficacy in inflammatory bowel disease. To investigate the effects of long-term administration of rifaximin on the liver, PXR-humanized mice were administered rifaximin for 6 months; wild-type and Pxr-null mice were treated in parallel as controls. Histological analysis revealed time-dependent intense hepatocellular fatty degeneration and increased hepatic triglycerides in PXR-humanized mice and not in wild-type and Pxr-null mice. After long-term treatment, PXR target genes were induced in small intestine and liver, with significant up-regulation in the expression of hepatic genes related to triglyceride synthesis and lipid accumulation. However, no significant hepatic accumulation of rifaximin was found, even after 6 months of treatment, in PXR-humanized mice. Genes in the small intestine that are involved in the uptake of fatty acids and triglycerides were induced along with increased triglyceride accumulation in intestinal epithelial cells of PXR-humanized mice; this was not observed in wild-type and Pxr-null mice. These findings suggest that long-term administration of rifaximin could lead to PXR-dependent hepatocellular fatty degeneration as a result of activation of genes involved in lipid uptake, thus indicating a potential adverse effect of rifaximin on liver function after long-term exposure.

Reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatin-mediated acute kidney injury

Peroxisome proliferator-activated receptor-α (PPARα) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPARα and CP on expression and enzyme activity of kidney lipoprotein lipase (LPL) as well as on expression of angiopoietin protein-like 4 (Angptl4), glycosylphosphatidylinositol-anchored-HDL-binding protein (GPIHBP1), and lipase maturation factor 1 (Lmf1), which are recognized as important proteins that modulate LPL activity. CP caused a 40% reduction in epididymal white adipose tissue (WAT) mass, with a reduction of LPL expression and activity. CP also reduced kidney LPL expression and activity. Angptl4 mRNA levels were increased by ninefold in liver and kidney tissue and by twofold in adipose tissue of CP-treated mice. Western blots of two-dimensional gel electrophoresis identified increased expression of a neutral pI Angptl4 protein in kidney tissue of CP-treated mice. Immunolocalization studies showed reduced staining of LPL and increased staining of Angptl4 primarily in proximal tubules of CP-treated mice. CP also increased TG accumulation in kidney tissue, which was ameliorated by PPARα ligand. In summary, a PPARα ligand ameliorates CP-mediated nephrotoxicity by increasing LPL activity via increased expression of GPHBP1 and Lmf1 and by reducing expression of Angptl4 protein in the proximal tubule.

Folate modulates adipocyte promoter methylation and lipid filling and release

Nutritional inputs may modify gene promoter methylation and thereby affect adipocyte metabolism. We differentiated 3T3L1 adipocytes under low, standard and enhanced folate levels followed by maturation under similar treatments plus the methyl donor S‐adenosylmethionine (SAM) and methylation inhibitor, 5‐aza‐2‐deoxycitidine (2DC). Folate exposure during differentiation dose‐dependently decreased triglyceride accumulation, lipolysis and proliferation (p<0.001) while folate exposure during maturation dose‐dependently increased triglyceride accumulation and lipolysis (p<0.001). Adipocytes differentiated under low folate conditions showed increased mRNA levels of key metabolic genes after exposure to high folate conditions during maturation ; hormone sensitive lipase (Lipe, +54.25%), fatty acid synthase (Fasn, +288%), glucose transporter 4 (Glut4,+141%), leptin (Lep, +450%). Adipocyte genomic DNA was hybridized against Nimblegen whole genome CpG promoter arrays and RNA on Affymetrix Mouse arrays. Low folate exposure during maturation decreased promoter methylatation in Lipe and carboxylesterase (CES) with increased gene expression relative to standard conditions (1.17 & 4.14 FC respectively). Thus, adipocyte exposure to methyl donors during and following differentiation modulates lipid accumulation, and low folate during differentiation may promote lipid filling.Grant Funding Source : Internal Award

S24 * S-ADENOSYLMETHIONINE IN THE PATHOGENESIS AND TREATMENT OF ALCOHOLIC LIVER DISEASE * S24.1 * THE ROLE OF ALTERED SAME METABOLISM IN ALCOHOL-INDUCED ER STRESS AND LIVER INJURY

S24 * S-ADENOSYLMETHIONINE IN THE PATHOGENESIS AND TREATMENT OF ALCOHOLIC LIVER DISEASE * S24.1 * THE ROLE OF ALTERED SAME METABOLISM IN ALCOHOL-INDUCED ER STRESS AND LIVER INJURY

Effects of antipsychotics with different weight gain liabilities on human in vitro models of adipose tissue differentiation and metabolism

Weight gain and metabolic abnormalities are serious side effects associated with the use of several second generation antipsychotics (SGA). The adipose tissue has been considered a direct SGA target involved in the development of these adverse effects. Recent studies, mainly using murine cells, have suggested that SGA increase both adipogenesis of preadipocytes and lipid accumulation in mature adipocytes. However, to date there has been little research comparing the effects of antipsychotics with different propensities to induce weight gain on human in vitro models of white adipose tissue neoformation and metabolism. The present study aimed to investigate the effects of antipsychotics either strongly associated with weight gain, such as the SGA clozapine and olanzapine, or not, such as the SGA ziprasidone and the classical antipsychotic haloperidol, on proliferation and adipocyte differentiation of human adipose-derived stem cells (ADSCs) and lipogenesis in human mature adipocytes. Whereas ziprasidone induced elevated levels of cell death during adipogenesis and could not be investigated further, we observed that clozapine, olanzapine and haloperidol had slight stimulatory effects on the transcriptional program of ADSCs adipogenesis. However, the observed changes in adipocyte-specific genes were not accompanied by a significant increase in triglyceride accumulation within differentiated adipocytes. Our data also showed that these three antipsychotics displayed inhibitory effects on the proliferation rates of undifferentiated ADSCs. Regarding mature adipocyte metabolism, we observed that olanzapine slightly inhibited insulin-stimulated lipogenesis at the highest concentration used, and haloperidol exerted the strongest inhibitory effects on both basal and insulin-stimulated lipogenesis. Taken together, our results suggest that a direct and potent effect of clozapine and olanzapine on adipose tissue biology is not an important mechanism by which these SGA induce metabolic disturbances in humans. On the other hand, the haloperidol-mediated downregulation of the lipogenic capacity of human adipose tissue may be a possible mechanism contributing to its lower propensity to induce serious metabolic side effects.

Fucoxanthin exerts differing effects on 3T3-L1 cells according to differentiation stage and inhibits glucose uptake in mature adipocytes

Progression of 3T3-L1 preadipocyte differentiation is divided into early (days 0–2, D0–D2), intermediate (days 2–4, D2–D4), and late stages (day 4 onwards, D4-). In this study, we investigated the effects of fucoxanthin, isolated from the edible brown seaweed Petalonia binghamiae, on adipogenesis during the three differentiation stages of 3T3-L1 preadipocytes. When fucoxanthin was applied during the early stage of differentiation (D0–D2), it promoted 3T3-L1 adipocyte differentiation, as evidenced by increased triglyceride accumulation. At the molecular level, fucoxanthin increased protein expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), sterol regulatory element-binding protein 1c (SREBP1c), and aP2, and adiponectin mRNA expression, in a dose-dependent manner. However, it reduced the expression of PPARγ, C/EBPα, and SREBP1c during the intermediate (D2–D4) and late stages (D4–D7) of differentiation. It also inhibited the uptake of glucose in mature 3T3-L1 adipocytes by reducing the phosphorylation of insulin receptor substrate 1 (IRS-1). These results suggest that fucoxanthin exerts differing effects on 3T3-L1 cells of different differentiation stages and inhibits glucose uptake in mature adipocytes.

Role for PPARγ in obesity‐induced hepatic steatosis as determined by hepatocyte‐ and macrophage‐specific conditional knockouts

Peroxisome proliferator-activated receptor (PPAR) γ is a nuclear receptor central to glucose and lipid homeostasis. PPARγ role in nonalcoholic fatty liver disease is controversial because PPARγ overexpression is a general property of steatotic livers, but its activation by thiazolidinediones reduces hepatic steatosis. Here, we investigated hepatic PPARγ function by using Cre-loxP technology to generate hepatocyte (PPARγ(Δhep))- and macrophage (PPARγ(Δmac))-specific PPARγ-knockout mice. Targeted deletion of PPARγ in hepatocytes, and to a lesser extent in macrophages, protected mice against high-fat diet-induced hepatic steatosis. Down-regulated expression of genes involved in lipogenesis (SCD1, SREBP-1c, and ACC), lipid transport (CD36/FAT, L-FABP, and MTP), and β-oxidation (PPARα and ACO) was observed in PPARγ(Δhep) mice. Moreover, PPARγ(Δhep) mice showed improved glucose tolerance and reduced PEPCK expression without changes in Pcx, Fbp1, and G6Pc expression and CREB and JNK phosphorylation. In precision-cut liver slices (PCLSs) and hepatocytes, rosiglitazone either alone or in combination with oleic acid increased triglyceride accumulation, an effect that was blocked by the PPARγ antagonist biphenol A diglycidyl ether (BADGE). PCLSs and hepatocytes from PPARγ(Δhep) mice showed blunted responses to rosiglitazone and oleic acid, whereas the response to these compounds remained intact in PCLSs from PPARγ(Δmac) mice. Collectively, these findings establish PPARγ expression in hepatocytes as a prosteatotic factor in fatty liver disease.

Cardiac triglyceride accumulation following acute lipid excess occurs through activation of a FoxO1–iNOS–CD36 pathway

Obesity due to nutrient excess leads to chronic pathologies including type 2 diabetes and cardiovascular disease. Related to nutrient excess, FoxO1 has a role in regulating fatty acid uptake and oxidation and triglyceride (TG) storage by mechanisms that are largely unresolved. We examined the mechanism behind palmitate (PA)-induced TG accumulation in cardiomyocytes. To mimic lipid excess, rat ventricular myocytes were incubated with albumin-bound PA (1mM) or rats were administered Intralipid (20%). PA-treated cardiomyocytes showed a substantial increase in TG accumulation, accompanied by amplification of nuclear migration of phospho-p38 and FoxO1, iNOS induction, and translocation of CD36 to the plasma membrane. PA also increased Cdc42 protein and its tyrosine nitration, thereby rearranging the cytoskeleton and facilitating CD36 translocation. These effects were duplicated by TNF-α and reversed by the iNOS inhibitor 1400W. PA increased the nuclear interaction between FoxO1 and NF-κB, reduced the nuclear presence of PGC-1α, and downregulated expression of oxidative phosphorylation proteins. In vivo a robust increase in cardiac TGs after Intralipid administration was also associated with augmentation of nuclear FoxO1 and iNOS expression. Impeding this FoxO1–iNOS–CD36 pathway could decrease cardiac lipid accumulation and oxidative/nitrosative stress and help ameliorate the cardiovascular complications associated with obesity and diabetes.

A mouse model for nonalcoholic steatohepatitis

Background Nonalcoholic steatohepatitis (NASH) is a hepatic manifestation of the growing metabolic syndrome epidemic that could progress to cirrhosis. Animal models adequately mimicking this condition in humans are scanty. Aim The objective of our study was to investigate whether high-fat diets (HFD) with adequate methionine and choline levels can induce pathophysiological features typical of human NASH in C57BL/6J mice. Methods Forty C57BL/6J mice, divided into control and high-fat (HF) groups, were fed low-fat diet and HFD, ad libitum respectively for 20 weeks. At the end of 20 weeks, animals were sacrificed and assays were performed for blood biomarkers typical of human NASH. Adipose tissue depots were collected and liver samples were processed for histological examination. Results High-fat feeding led to increased triglyceride accumulation in the liver (8.9 μmol/100 mg liver tissue vs. 2.6 μmol/100 mg for control) and induced histopathological features of human NASH including hepatic steatosis, ballooning inflammation and fibrosis. Expressions of proteins and chemokines predominant in NASH including collagens I, III and IV and platelet derived growth factor (PDGF) A and B were significantly higher in animals fed the HFD. Liver enzymes alanine transaminase, aspartate transaminase and alkaline phosphatase were significantly ( P<.05) elevated in the HF group compared to controls. Mice on HFD also developed hyperglycemia, hyperinsulinemia, hypoadiponectinemia along with elevated tumor necrosis factor α, resistin, leptin, free fatty acids, transforming growth factor β and malondialdehyde levels that characterize NASH in humans. Conclusion Long-term HF feeding with adequate methionine and choline can induce many of the pathophysiological features typical of human NASH in C57BL/6J mice.

Identification of the lipid droplet targeting domain of the Cidea protein

Cidea, the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) domain-containing protein, is targeted to lipid droplets in mouse adipocytes, where it inhibits triglyceride hydrolysis and promotes lipid storage. In mice, Cidea may prevent lipolysis by binding and shielding lipid droplets from lipase association. Here we demonstrate that human Cidea localizes with lipid droplets in both adipocyte and nonadipocyte cell lines, and we ascribe specific functions to its protein domains. Expression of full-length Cidea in undifferentiated 3T3-L1 cells or COS-1 cells increases total cellular triglyceride and strikingly alters the morphology of lipid droplets by enhancing their size and reducing their number. Remarkably, both lipid droplet binding and increased triglyceride accumulation are also elicited by expression of only the carboxy-terminal 104 amino acids, indicating this small domain directs lipid droplet targeting and triglyceride shielding. However, unlike the full-length protein, expression of the carboxy-terminus causes clustering of small lipid droplets but not the formation of large droplets, identifying a novel function of the N terminus. Furthermore, human Cidea promotes lipid storage via lipolysis inhibition, as the expression of human Cidea in fully differentiated 3T3-L1 adipocytes causes a significant decrease in basal glycerol release. Taken together, these data indicate that the carboxy-terminal domain of Cidea directs lipid droplet targeting, lipid droplet clustering, and triglyceride accumulation, whereas the amino terminal domain is required for Cidea-mediated development of enlarged lipid droplets.

Effects of Hyperinsulinemia on Lipolytic Function of Three-Dimensional Adipocyte/Endothelial Co-Cultures

The increased incidence of both type 2 diabetes mellitus and obesity has prompted the need to develop physiologically relevant adipose tissue models for controlled study of both normal and diseased adipose functions. Insulin resistance, characteristic of both type 2 diabetes mellitus and obesity, is often preceded by hyperinsulinemia. We propose here a three-dimensional (3D) co-culture adipose tissue model to study the effects of high insulin exposure, which enabled the study of physiological cell responses to hyperinsulinemic conditions. Two-dimensional adipocyte studies were initially conducted to establish a baseline control in which insulin levels were established. Adipocytes and endothelial cells were subsequently co-cultured on 3D porous silk fibroin scaffolds in normal or high insulin concentrations, and their physiological responses were assessed with respect to lipogenesis and lipolysis. High insulin levels stimulated both an increase in triglyceride accumulation and a decrease in lipolysis levels compared to that of normal insulin conditions. In contrast, adipocyte monocultures did not exhibit any differences between insulin levels. The ability of this 3D system to elicit physiological responses to hyperinsulinemia in co-culture serves as a significant step forward in adipose tissue engineering. The development of physiologically relevant 3D in vitro adipose tissue models presents promise for the study of disease mechanisms as well as in assessing therapeutic treatments.

Role of tumor necrosis factor α (TNFα) in the onset of fructose-induced nonalcoholic fatty liver disease in mice

Tumor necrosis factor α (TNFα) is known to be involved in dysregulation of hepatic lipid metabolism and insulin signaling. However, whether TNFα also plays a casual role in the onset of fructose-induced nonalcoholic fatty liver disease (NAFLD) has not yet been determined. Therefore, wild-type and TNFα receptor 1 (TNFR1)−/− mice were fed with either 30% fructose solution or plain tap water. Hepatic triglycerides, markers of inflammation and ATP concentration as well as plasma ALT levels were determined. Hepatic PAI-1, SREBP-1, FAS mRNA expression was assessed by real-time RT-PCR. Furthermore, lipid peroxidation and indices of insulin resistance were determined in liver tissue and plasma. In comparison to water controls, chronic intake of 30% fructose solution caused a significant ∼5-fold increase in triglyceride accumulation and neutrophil infiltration in livers of wild-type mice and a ∼8-fold increase in plasma ALT levels. In TNFR1−/− mice, hepatic steatosis was attenuated and neutrophil infiltration in the liver as well as plasma ALT levels was similar to water controls. The protective effect of the TNFR1 deletion against the onset of fructose-induced steatosis was associated with increased phospho AMPK and Akt levels, decreased SREBP-1 and FAS expression in the liver and decreased RBP4 plasma levels, whereas hepatic lipid peroxidation, iNOS protein and ATP levels were similar between wild-type and TNFR1−/− mice fed fructose. Taken together, these data suggest that TNFα plays a casual role in the onset of fructose-induced liver damage as well as insulin resistance in mice through signaling cascades downstream of TNFR1.

A New Role for GPR103b in the Peripheral Regulation of Adipogenesis

The activation of G protein-coupled receptor 103 (GPR103) by its endogenous peptidic ligands, QRFPs, is involved in the central regulation of feeding by increasing food intake, body weight, and fat mass after intracerebroventricular injection in mice. However, the role of GPR103 in regulating peripheral metabolic pathways has not yet been explored. The present study aimed to investigate the role of GPR103 in adipogenesis and lipid metabolism using 3T3-L1 adipocyte cells. Our results show that differentiated 3T3-L1 cells expressed the GPR103b subtype mRNA and protein, as well as QRFP mRNA. QRFP-43 and 26 induced an increase in triglyceride accumulation of 50 and 41%, respectively, and elicited a dose-dependent increase in fatty acid uptake, by up to approximately 60% at the highest concentration, in 3T3-L1-differentiated cells. QRFP-43 and 26 inhibited isoproterenol (ISO)-induced lipolysis in a dose-dependent manner, with IC50s of 2.3 1.2 and 1.1 1.0 nM, respectively. The expression of genes involved in lipid uptake (FATP1, CD36, LPL, ACSL1, PPAR, and C/EBP), was increased by 2to 3-fold following treatment with QRFP. The effects of QRFP on ISO-induced lipolysis and fatty acid uptake were abolished when GPR103b was silenced. In a mouse model of diet-induced obesity, the expression of GPR103b in epididymal fat pads was elevated by 16-fold whereas that of QRFP was reduced by 46% compared to lean mice. Furthermore, QRFP was bioactive in omental adipocytes from obese individuals, inhibiting ISO-induced lipolysis in these cells. Our results suggest that GPR103b and QRFP work in an autocrine/paracrine manner to regulate adipogenesis. Foxa1 and Foxa2 Maintain the Metabolic and Secretory Features of the Mature -Cell Nan Gao, John Le Lay, Wei Qin, Nicolai Doliba, Jonathan Schug, Alan J. Fox, Olga Smirnova, Franz M. Matschinsky, and Klaus H. Kaestner (Mol Endocrinol, published June 9, 2010, 10.1210/me.2009-0513) ABSTRACT Foxa1 and Foxa2 play both redundant and distinct roles in early pancreas development. We demonstrate here that inducible ablation of both transcription factors in mature mouse -cells leads to impaired glucose homeostasis and insulin secretion. The defects in both glucose-stimulated insulin secretion and intracellular calcium oscillation are more pronounced than those in -cells lacking only Foxa2. Unexpectedly, in contrast to the severe reduction of -cell-enriched factors contributing to metabolic and secretory pathways, expression of a large number of genes that are involved in neural differentiation and function is significantly elevated. We further demonstrate that expression of carbohydrate response element-binding protein (ChREBP or Mlxipl), an important transcriptional regulator of carbohydrate metabolism, is significantly affected in compound Foxa1/a2 mutant -cells. ChREBP expression is directly controlled by Foxa1 and Foxa2 in both the fetal endocrine pancreas as well as mature islets. These data demonstrate that Foxa1 and Foxa2 play crucial roles in the development and maintenance of -cell-specific secretory and metabolic pathways. 3564 Abstracts Translational Highlights from MOLECULAR ENDOCRINOLOGY J Clin Endocrinol Metab, July 2010, 95(7):3563–3564Foxa1 and Foxa2 play both redundant and distinct roles in early pancreas development. We demonstrate here that inducible ablation of both transcription factors in mature mouse -cells leads to impaired glucose homeostasis and insulin secretion. The defects in both glucose-stimulated insulin secretion and intracellular calcium oscillation are more pronounced than those in -cells lacking only Foxa2. Unexpectedly, in contrast to the severe reduction of -cell-enriched factors contributing to metabolic and secretory pathways, expression of a large number of genes that are involved in neural differentiation and function is significantly elevated. We further demonstrate that expression of carbohydrate response element-binding protein (ChREBP or Mlxipl), an important transcriptional regulator of carbohydrate metabolism, is significantly affected in compound Foxa1/a2 mutant -cells. ChREBP expression is directly controlled by Foxa1 and Foxa2 in both the fetal endocrine pancreas as well as mature islets. These data demonstrate that Foxa1 and Foxa2 play crucial roles in the development and maintenance of -cell-specific secretory and metabolic pathways. 3564 Abstracts Translational Highlights from MOLECULAR ENDOCRINOLOGY J Clin Endocrinol Metab, July 2010, 95(7):3563–3564

GPR103b Functions in the Peripheral Regulation of Adipogenesis

The activation of G protein-coupled receptor 103 (GPR103) by its endogenous peptidic ligands, QRFPs, is involved in the central regulation of feeding by increasing food intake, body weight, and fat mass after intracerebroventricular injection in mice. However, the role of GPR103 in regulating peripheral metabolic pathways has not yet been explored. The present study aimed to investigate the role of GPR103 in adipogenesis and lipid metabolism using 3T3-L1 adipocyte cells. Our results show that differentiated 3T3-L1 cells expressed the GPR103b subtype mRNA and protein, as well as QRFP mRNA. QRFP-43 and -26 induced an increase in triglyceride accumulation of 50 and 41%, respectively, and elicited a dose-dependent increase in fatty acid uptake, by up to approximately 60% at the highest concentration, in 3T3-L1-differentiated cells. QRFP-43 and -26 inhibited isoproterenol (ISO)-induced lipolysis in a dose-dependent manner, with IC(50)s of 2.3 +/- 1.2 and 1.1 +/- 1.0 nm, respectively. The expression of genes involved in lipid uptake (FATP1, CD36, LPL, ACSL1, PPAR-gamma, and C/EBP-alpha), was increased by 2- to 3-fold after treatment with QRFP. The effects of QRFP on ISO-induced lipolysis and fatty acid uptake were abolished when GPR103b was silenced. In a mouse model of diet-induced obesity, the expression of GPR103b in epididymal fat pads was elevated by 16-fold whereas that of QRFP was reduced by 46% compared to lean mice. Furthermore, QRFP was bioactive in omental adipocytes from obese individuals, inhibiting ISO-induced lipolysis in these cells. Our results suggest that GPR103b and QRFP work in an autocrine/paracrine manner to regulate adipogenesis.

Suppression of Extracellular Signal‐regulated Kinase‐1/2 Causes Fat Accumulation in Hepatocytes: A Potential Mechanism in Alcoholic Fatty Liver Disease (ALD)

Although much progress has been made, the underlying mechanism in alcohol‐induced hepatic steatosis is still obscure. In this study, we examined the effect of alcohol exposure on MEK/ERK1/2 activation and its potential involvement in the development of hepatic steatosis. In animal studies, male C57BL/6 mice were pair‐fed with liquid diets containing either ethanol or an isocaloric maltose‐dextrin mixture for four weeks. In the in vitro investigations, HepG2 cells were utilized. Our results showed that chronic alcohol feeding resulted in increased triglyceride accumulation in the liver and liver injury in mice. These observations were concomitant with decreased S‐adenosylmethionine/S‐adenosylhomocysteine ratio and suppressed ERK 1/2 activation in the liver. Using HepG2 cells, we found that ERK inhibition by U0126 elevated intracellular triglyceride contents, and this was associated with increased gene expression of diacylglycerol acyltransferase‐2 (DGAT2). In contrast, epidermal growth factor, a major activator of ERK1/2, reduced triglyceride synthesis. Furthermore, N‐acetyl‐S‐farnesyl‐L‐cysteine (AFC), a methyltrasferase inhibitor, elevated triglyceride synthesis, which is associated with ERK ½ inhibition and enhanced DGAT2 expression. Taken together, our findings suggest that suppression of ERK‐1/2 activation may play a mechanistic role in the development of ALD.Supported by the National Institutes of Health grants K01 AA015344 and R01 AA017442 (Z Song).

A mouse model for Non‐alcoholic Steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a hepatic manifestation of metabolic syndrome that could progress to cirrhosis. Animal models adequately mimicking this condition in humans are scanty. The main objective of this study was to investigate whether high‐fat diets (HFD) with adequate methionine and choline levels can induce pathophysiological features typical of human NASH in C57BL/6J mice. Forty C57BL/6J mice, divided into control and high‐fat groups were fed low‐fat diet or HFD ad libitum for 20 weeks. At the end of 20 weeks animals were sacrificed and assays were performed for blood biomarkers typical of human NASH. HFD led to increased triglyceride accumulation in the liver and induced histopathological features of human NASH including hepatic steatosis, ballooning, inflammation and fibrosis. Liver enzymes alanine transaminase (ALT‐80.12 IU/L), aspartate transaminase (AST‐ 64.23 IU/L) and alkaline phosphatase (AP ‐ 67.43 IU/L) were significantly elevated in the high‐fat group compared to controls (ALT ‐ 38.14 IU/L; AST ‐ 23.18 IU/L, and AP ‐ 25.05 IU/L). Mice on HFD also developed hyperglycemia, hyperinsulinemia, hypoadiponectinemia along with elevated tumor necrosis factor (TNF‐α), resistin, hs‐CRP, free fatty acids, and malondialdehyde levels that characterize NASH in humans. Long‐term HF feeding with adequate methionine and choline can induce many of the pathophysiological features of human NASH in C57BL/6J mice.Supported by Research Enhancement Program, TWU and Texas Department of Agriculture

High‐fat diet with adequate methionine and choline levels induces hepatic insulin resistance via Akt signaling pathway in a rodent model for Non‐alcoholic Steatohepatitis.

Signaling pathways involved in hepatic insulin resistance that develops in animal models of non‐alcoholic steatohepatitis (NASH) have not been adequately investigated. In this study hepatic insulin resistance was investigated in livers of C57BL/6J mice by determining protein and gene expressions of insulin receptor (IR), insulin receptor substrate (IRS), Akt and their phosphorylated forms (p‐IR, p‐IRS, and p‐Akt). Forty C57BL/6J mice, divided into control and high‐fat groups were fed low‐fat diet or HFD ad libitum for 20 weeks. At the end of 20 weeks animals were sacrificed and assays were performed for blood biomarkers typical of human NASH. As reported earlier, HFD led to increased triglyceride accumulation in the liver and induced histopathological features of human NASH including hepatic steatosis, ballooning, inflammation and fibrosis. At the end of 20 weeks, ratio of p‐IR/IR, p‐IRS/IRS, and p‐Akt/Akt expressions were significantly lower in the HF group than controls along with decreased expression's of total and phosphorylated forms. Significant reductions in the gene expressions of IR, IRS‐2, Akt and their phosphorylated forms were observed in the HF group compared to controls. Thus, hepatic insulin resistance in this NASH model is mediated via Akt signaling pathway.Supported by Research Enhancement Program, TWU and Texas Department of Agriculture

The role of insulin and glucose in goose primary hepatocyte triglyceride accumulation

In order to obtain some information on how fatty liver arises in geese, we investigated the role of insulin and glucose in triglyceride (TG) accumulation in goose primary hepatocytes. Goose primary hepatocytes were isolated and treated with insulin and glucose. Compared with the control group, 100 and 150 nmol l(-1) insulin increased TG accumulation, acetyl-CoA carboxylase-alpha (ACCalpha) and fatty acid synthase (FAS) activity, and the mRNA levels of sterol regulatory element-binding protein-1 (SREBP-1), FAS and ACCalpha genes. Insulin at 200 nmol l(-1) had an inhibiting effect on TG accumulation and the activity of ACC and FAS, but increased the gene expression of SREBP-1, FAS and ACCalpha. We also found that high glucose (30 mmol l(-1)) increased the TG level, ACC and FAS activity, and the mRNA levels of SREBP-1 and FAS. However, there was no effect of high glucose on ACCalpha mRNA level. In addition, the interaction between insulin and glucose was observed to induce TG accumulation, ACC and FAS activity, and gene expression of SREBP-1, FAS and ACCalpha, and increase SREBP-1 nuclear protein level and binding of nuclear SREBP-1 and the SRE response element of the ACC gene. The result also indicated that the glucose-induced TG accumulation decreased after 96 h when the hepatocytes were cultured with 30 mmol l(-1) glucose. In conclusion, insulin and glucose may affect hepatic lipogenesis by regulating lipogenic gene expression and lipogenic enzyme activity in goose hepatocytes, and SREBP-1 might play an important role in the synergetic activation of lipogenic genes. We propose that the utilization of accumulated TG in hepatocytes is the reason for the reversible phenomenon in goose hepatocellular steatosis.

MicroRNAs Induced During Adipogenesis that Accelerate Fat Cell Development Are Downregulated in Obesity

OBJECTIVEWe investigated the regulation and involvement of microRNAs (miRNAs) in fat cell development and obesity.RESEARCH DESIGN AND METHODSUsing miRNA microarrays, we profiled the expression of >370 miRNAs during adipogenesis of preadipocyte 3T3-L1 cells and adipocytes from leptin deficient ob/ob and diet-induced obese mice. Changes in key miRNAs were validated by RT-PCR. We further assessed the contribution of the chronic inflammatory environment in obese adipose tissue to the dysregulated miRNA expression by tumor necrosis factor (TNF)-α treatment of adipocytes. We functionally characterized two adipocyte-enriched miRNAs, miR-103 and miR-143, by a gain-of-function approach.RESULTSSimilar miRNAs were differentially regulated during in vitro and in vivo adipogenesis. Importantly, miRNAs that were induced during adipogenesis were downregulated in adipocytes from both types of obese mice and vice versa. These changes are likely associated with the chronic inflammatory environment, since they were mimicked by TNF-α treatment of differentiated adipocytes. Ectopic expression of miR-103 or miR-143 in preadipocytes accelerated adipogenesis, as measured both by the upregulation of many adipogenesis markers and by an increase in triglyceride accumulation at an early stage of adipogenesis.CONCLUSIONSOur results provide the first experimental evidence for miR-103 function in adipose biology. The remarkable inverse regulatory pattern for many miRNAs during adipogenesis and obesity has important implications for understanding adipose tissue dysfunction in obese mice and humans and the link between chronic inflammation and obesity with insulin resistance.

Long-chain Polyunsaturated Fatty Acid Transport across Human Placental Choriocarcinoma (BeWo) Cells

Long-chain polyunsaturated fatty acids (LCPUFAs) such as docosahexaenoic acid (DHA) and arachidonic acid (AA) are essential for proper development of fetal brain and retina. These LCPUFAs are selectively enriched in the fetal circulation compared with the maternal circulation. In the current study we investigated the transfer of LCPUFAs and a non-essential fatty acid (oleic acid, OA) in a transwell monolayer system of placental choriocarcinoma (BeWo) cells. We show that incubation with OA results in increased triglyceride accumulation and lipid droplet formation compared with that of DHA. The relative amount of transfer of DHA across the cell monolayer was ∼4-fold greater compared with that of OA when these fatty acids were added individually at 100 μM. This reflects the different fates of these two fatty acids in their metabolism and subsequent transport across the placental trophoblasts to the fetus. When using a mixture of fatty acids mimicking the composition of plasma non-esterified fatty acids during the last trimester of pregnancy, the transfer of OA and the LCPUFAs (DHA and AA) into the basolateral reservoir was not significantly different, whereas the transfer of palmitic acid (PA) was ∼3.5-fold higher than OA transfer. However, since the concentration of OA compared to LCPUFAs was 10-fold higher in the donor chamber, the relative transport of the LCPUFAs was higher compared with that of OA. In addition, we show that inhibiting esterification of fatty acids into acyl-CoA can modulate, in part, the degree of transport through the cells. In conclusion, the transwell model system closely mimics the mechanisms of differential fatty acid transport as observed in vivo. LCPUFAs were transported through the cells more efficiently than shorter fatty acids such as OA.