▼ Dietary phytosterol supplements are readily available to consumers since they effectively reduce plasma low-density lipoprotein cholesterol. Several studies on cell cultures and xenograft mouse models suggest that dietary phytosterols may also exert protective effects against common cancers. We examined the effects of a dietary phytosterol supplement on tumor onset and progression using the well-characterized mouse mammary tumor virus polyoma virus middle T antigen transgenic mouse model of inherited breast cancer. Both the development of mammary hyperplastic lesions (at age 4 weeks) and total tumor burden (at age 13 weeks) were reduced after dietary phytosterol supplementation in female mice fed a high-fat, high-cholesterol diet. A blind, detailed histopathologic examination of the mammary glands (at age 8 weeks) also revealed the presence of less-advanced lesions in phytosterol-fed mice. This protective effect was not observed when the mice were fed a low-fat, low-cholesterol diet. Phytosterol supplementation was effective in preventing lipoprotein oxidation in mice fed the high-fat diet, a property that may explain - at least in part - their anticancer effects since lipoprotein oxidation/inflammation has been shown to be critical for tumor growth. In summary, our study provides preclinical proof of the concept that dietary phytosterols could prevent the tumor growth associated with fat-rich diet consumption.

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To understand the molecular mechanisms underlying alterations in the pathophysiologic status of dietary obesity, we examined hepatic genes differentially expressed in a long-term high-fat intake-induced obesity mouse model. C57BL/6J male mice were fed with two kinds of diets for 12 weeks; a low-fat diet (LFD), a high-fat diet (HFD; n=8), and the expression levels of ∼10,000 transcripts in liver tissues from the two groups were assessed using cDNA microarray analysis. Twelve-week feeding with the HFD resulted in significant increase in body weight, visceral fat accumulation and circulating cholesterol concentration, compared with the LFD group. The cDNA microarray analysis revealed marked differences in the expressions of 97 hepatic genes. These genes were categorized into seven groups:

1. metabolism;

2. defense, stress, and inflammation responses;

3. signal transduction, apoptosis, and cell cycle;

4. transcription regulation;

5. protein synthesis and modification;

6. transport; and

7. cellular adhesion, cytoskeleton and trafficking.

The expression of genes involved in fatty acid catabolism and ketone body synthesis, such as acyl–CoA oxidase1 (Acox1) and HMG–CoA lyase (Hmgcl), was significantly increased, and expression of genes involved in lipogenesis and cholesterol synthesis, such as acetyl–CoA synthetase2 (Acs2), fatty acid synthase (Fasn), and squalene epoxidase (Sqle), was drastically decreased in the HFD group. Interestingly, the genes implicated in defense and stress responses, such as glutathione S-transferases (GSTs) and heat shock proteins (Hsps), were also highly represented in the HFD group. Besides, a number of previously unappreciated regulatory molecules were changed by the HFD. These results revealed a transcriptional adaptation to long-term HFD and provided interesting information about the molecules involved in the development and maintenance of the obesity phenotype in vivo.

▼ Parkinson disease is a neurodegenerative pathology that has been linked to several genetic mutations of the SNCA gene encoding the pro-oxidant α-synuclein protein. The budding yeast Saccharomyces cerevisiae is a valuable model for studying the cellular and molecular mechanisms of α-synuclein toxicity. Indeed heterologous expression of α-synuclein is toxic to wild-type yeast and exhibits the main features of damage caused to mammalian neurons, including an increase in neutral lipid storage (triglycerides and steryl esters, embedded into lipid droplets). To address the significance of this accumulation, we forced α-synuclein production in a strain unable to synthesize triglycerides and steryl esters. Surprisingly, the inability to store neutral lipids rendered the cells more tolerant to α-synuclein. Our results indicate that the level of α-synuclein toxicity is correlated with fatty acid synthase activity and intracellular redox status.

▼ In the past years, free fatty acids (FFAs) and obesity have been reported to play an important role in cancer development. Palmitic acid (PA) is the most prevalent saturated FFA in circulation. However, the mechanism underlying the effect of PA on cell proliferation is still to be elucidated. In this report, we, for the first time, investigate the signaling pathway in human normal hepatocytes (QZG) responsible for PA-induced proliferation. The results demonstrate that PA promotes cell cycle progression, accelerates cell proliferation, and induces a transient and sequential activation of a series of kinases. The employment of several inhibitors and antioxidants indicates that a ROS-induced stress-sensitive p38 MAPK/ERK-Akt cascade plays a critical role in the regulation of PA on cell cycle and cell proliferation. Moreover, PA dose and time dependently activates Nrf2 and this activation relies on ROS-induced stimulation of p38 MAPK/ERK-Akt signaling, demonstrating that Nrf2 activation may be associated with the regulation of PA on cell cycle transition and proliferation. In conclusion, our study elucidates the importance of PA metabolism on cell proliferation, and suggests that PA stimulates hepatocyte proliferation through activating the ROS-p38 MAPK/ERK-Akt cascade which is intersected with the activation of Nrf2 and that the effect of ROS on signal transduction is in a dose- and time-dependent manner. All the above noted provide a new clue for the central role of ROS in cell proliferation and tumorigenesis.

▼ In the past years, free fatty acids (FFAs) and obesity have been reported to play an important role in cancer development. Palmitic acid (PA) is the most prevalent saturated FFA in circulation. However, the mechanism underlying the effect of PA on cell proliferation is still to be elucidated. In this report, we, for the first time, investigate the signaling pathway in human normal hepatocytes (QZG) responsible for PA-induced proliferation. The results demonstrate that PA promotes cell cycle progression, accelerates cell proliferation, and induces a transient and sequential activation of a series of kinases. The employment of several inhibitors and antioxidants indicates that a ROS-induced stress-sensitive p38 MAPK/ERK-Akt cascade plays a critical role in the regulation of PA on cell cycle and cell proliferation. Moreover, PA dose and time dependently activates Nrf2 and this activation relies on ROS-induced stimulation of p38 MAPK/ERK-Akt signaling, demonstrating that Nrf2 activation may be associated with the regulation of PA on cell cycle transition and proliferation. In conclusion, our study elucidates the importance of PA metabolism on cell proliferation, and suggests that PA stimulates hepatocyte proliferation through activating the ROS-p38 MAPK/ERK-Akt cascade which is intersected with the activation of Nrf2 and that the effect of ROS on signal transduction is in a dose- and time-dependent manner. All the above noted provide a new clue for the central role of ROS in cell proliferation and tumorigenesis.

▼ Metabolic adaptations are triggered in the maternal organism to synthesize milk with an adequate concentration of long-chain polyunsaturated fatty acids (LC-PUFAs) required to the newborn. They may be a high uptake of dietary linoleic acid and its conversion to LC-PUFAs by desaturases of fatty acids (FADS) 1 and 2 in the mammary gland (MG). It is unknown if they also occur from onset of pregnancy. The aim of this study was to explore the participation of the MG as a mechanism involved in LC-PUFAs synthesis to support their demand during pregnancy and lactation in rats. The expression of desaturases in MG was significantly (P<0.05) higher (12.3-fold for FADS1 and 41.2-fold for FADS2) during the late pregnancy and throughout lactation (31.7-fold for FADS1 and 67.1-fold higher for FADS2) than in nonpregnant rats. SREBF-1c showed a similar pattern of increase during pregnancy but remained higher only during the early lactation (11.7-fold, P<0.005). Transcript of ELOVL6 and FASN increased throughout pregnancy and lactation, respectively. ELOVL5 mRNA increased in MG only during lactation (2.8 to 5.3-fold, P<0.005). Accordingly, a higher content of LC-PUFAs was found in lactating MG than in nonpregnant rats. Results suggest that MG participates from late pregnancy and throughout lactation by expressing desaturases and elongases as a mechanism involved in LC-PUFAs synthesis, probably by SREBF-1c. Because desaturases and ELOVL5 were expressed in cultured lactocytes and such expression was downregulated by linoleic and arachidonic acid, these cells may be a useful model for understanding the regulatory mechanisms for LC-PUFAs synthesis in MG.

▼ Metabolic adaptations are triggered in the maternal organism to synthesize milk with an adequate concentration of long-chain polyunsaturated fatty acids (LC-PUFAs) required to the newborn. They may be a high uptake of dietary linoleic acid and its conversion to LC-PUFAs by desaturases of fatty acids (FADS) 1 and 2 in the mammary gland (MG). It is unknown if they also occur from onset of pregnancy. The aim of this study was to explore the participation of the MG as a mechanism involved in LC-PUFAs synthesis to support their demand during pregnancy and lactation in rats. The expression of desaturases in MG was significantly (P<0.05) higher (12.3-fold for FADS1 and 41.2-fold for FADS2) during the late pregnancy and throughout lactation (31.7-fold for FADS1 and 67.1-fold higher for FADS2) than in nonpregnant rats. SREBF-1c showed a similar pattern of increase during pregnancy but remained higher only during the early lactation (11.7-fold, P<0.005). Transcript of ELOVL6 and FASN increased throughout pregnancy and lactation, respectively. ELOVL5 mRNA increased in MG only during lactation (2.8 to 5.3-fold, P<0.005). Accordingly, a higher content of LC-PUFAs was found in lactating MG than in nonpregnant rats. Results suggest that MG participates from late pregnancy and throughout lactation by expressing desaturases and elongases as a mechanism involved in LC-PUFAs synthesis, probably by SREBF-1c. Because desaturases and ELOVL5 were expressed in cultured lactocytes and such expression was downregulated by linoleic and arachidonic acid, these cells may be a useful model for understanding the regulatory mechanisms for LC-PUFAs synthesis in MG.

▼ Background/Aims

Resveratrol, a polyphenolic activator of the silent information regulation 2 homolog 1 (SIRT1), is known to extend lifespan and improve metabolic disease. The aim of the present study is to test whether resveratrol protects against metabolic steatohepatitis through the modulation of lipid metabolism-related genes.

Methods

We used a mouse model in which steatohepatitis can be induced by an atherogenic diet (Ath diet) to evaluate the effects of resveratrol on steatotic hepatitis and hepatic gene expression.

Results

The Ath diet induced excessive weight gain, hepatomegaly, dyslipidemia, and steatohepatitis after 8 weeks. The addition of resveratrol protected against Ath diet-induced changes and also alleviated steatohepatitis. Whole-genome expression analysis revealed that an Ath diet altered the hepatic expression of genes involved in lipid metabolism, and the addition of resveratrol to the diet reversed that effect. Real-time PCR and Western blot analysis confirmed the Ath diet up-regulated the levels of genes related to lipogenesis and down-regulated genes involved in lipolysis. Resveratrol clearly suppressed the Ath diet-induced alterations of the expression of genes related to lipid metabolism.

Conclusions

Resveratrol ameliorated dyslipidemia and steatohepatitis induced by the Ath diet, and its beneficial effects were associated with the altered expression of hepatic genes involved in lipid metabolism.

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Obesity has emerged as a global health problem with more than 1.1 billion adults to be classified as overweight or obese, and is associated with type 2 diabetes, cardiovascular disease, and several cancers. Since obesity is characterized by an increased size and/or number of adipocytes, elucidating the molecular events governing adipogenesis is of utmost importance. Recent findings indicate that microRNAs (miRNAs) – small non-protein-coding RNAs that function as post-transcriptional gene regulators – are involved in the regulatory network of adipogenesis. Whereas only a single human miRNA is known so far to be functional in adipogenesis as pro-adipogenic, several mouse miRNAs have been identified very recently as adipogenic regulators, thereby stimulating demand for studying the functional role of miRNAs during adipogenesis in human. Here, we demonstrate that miR-27b abundance decreased during adipogenesis of human multipotent adipose-derived stem (hMADS) cells. Overexpression of miR-27b blunted induction of PPARγ and C/EBPα, two key regulators of adipogenesis, during early onset of adipogenesis and repressed adipogenic marker gene expression and triglyceride accumulation at late stages. PPARγ has a predicted and highly conserved binding site in its 3′UTR and was indeed confirmed to be a direct target of miR-27b. Thus, these results suggest that the anti-adipogenic effect of miR-27b in hMADS cells is due, at least in part, to suppression of PPARγ.

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Peroxisome deficiency in liver causes hepatosteatosis both in patients and in mice. Here, we studied the mechanisms that contribute to this lipid accumulation and to activation of peroxisome proliferator activated receptor α (PPARα) by using liver-specific Pex5^−/− mice (L-Pex5^−/− mice). Surprisingly, steatosis was accompanied both by increased mitochondrial β-oxidation capacity, confirming previous observations, and by impaired de novo lipid synthesis mediated by reduced expression of sterol regulatory element binding protein 1c and its targets. As a consequence, when challenged with a high fat diet, L-Pex5^−/− mice were protected from adiposity. Hepatic fatty acid uptake was strongly increased whereas the expression of apolipoproteins and the lipoprotein assembly factor microsomal triglyceride transfer protein were markedly reduced resulting in reduced secretion of very low density lipoproteins. Most of these changes seemed to be orchestrated by the endogenous activation of PPARα, challenging the assumption that PPARα activation in hepatocytes requires fatty acid synthase dependent de novo fatty acid synthesis. Expression of cholesterol synthesizing enzymes and cholesterol levels were not affected in peroxisome deficient liver. In conclusion, increased fatty acid uptake driven by endogenous PPARα activation and reduced fatty acid secretion cause hepatosteatosis in peroxisome deficient livers.

▼ In the liver, maintaining lipid homeostasis is regulated by physiological and exogenous factors. These lipids are synthesized by Fasn, elongases and desaturases. Interactions in an organism among these factors are quite complex and, to date, relatively little is known about them. The aim of this study was to evaluate the coexisting role of physiological (insulin, fasting and feeding) and exogenous (dietary lipids) factors in the control of gene expression of Fasn, elongases and desaturases via Srebf-1c in liver from rats. Gene expression of encoding enzymes for fatty acid synthesis and fatty acid composition was evaluated in liver from rats in fasting and feeding (at 30, 60, 90 and 120min after feeding) when food intake (adequate or high-lipid diet) was synchronized to a restricted period of 7h. Fasn, Scd and Fads2 were induced during 120min after initial feeding in both dietary groups. This induction may be activated in part by insulin via Srebf-1c. Also, we showed for the first time that Elovl7 may be regulated by insulin and dietary lipids. The failure to synthesize saturated and monounsaturated fatty acids is consistent with a downregulation of Fasn and Scd, respectively, by dietary lipids. A higher content of LC-PUFAs was observed due to a high expression of Elovl2 and Elovl5, although Fads2 was suppressed by dietary lipids. Therefore, elongases may have a mechanism that is Srebf-1c-independent. This study suggests that a high-lipid diet triggers, during 120min after initial feeding, a tight coordination among de novo lipogenesis, elongation, and desaturation and may not always be regulated by Srebf-1c. Finally, upregulation by feeding (insulin) of Fasn, Scd, Fads2 and Srebf-1c is insufficient to compensate for the inhibitory effect of dietary lipids.

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Fatty acid-induced damage in pancreatic β-cells is assumed to play an important role in the development of type 2 diabetes. Lactogens (prolactin, placental lactogen and growth hormone) improve β-cell survival via STAT5 activation but the molecular targets are incompletely characterized. The aim of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic β-cells, and to examine this in relation to β-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP2, FATP1 and FATP4 were unchanged. RNAi against FAT/CD36 decreased fatty acid-induced apoptosis. Over-expression of constitutively active STAT5 was able to mimic hGH’s suppression of FAT/CD36 expression, whereas dominant negative STAT5 was unable to block the effect of hGH indicating that STAT5 did not bind directly to the FAT/CD36 promoter. The hGH-mediated suppression of FAT/CD36 mRNA was associated with a decrease in palmitate uptake and fatty acid-induced basal hyper-secretion of insulin resulting in improved glucose-stimulated insulin secretion. This study suggests that hGH can protect β-cells against fatty acid-induced damages.

▼ The objective of this study was to gain insight into the underlying mechanisms of fat deposition. Two sheep breeds with large fat-tail (Lori-Bakhtiari) and with thin-tail (Zel) were used as models. To determine important and key candidate lipid metabolism related genes, comparative genomic approaches were employed. Gene expression profiles of adipose tissues were analyzed in human, pig, and cattle by express sequence tag (EST) analysis. EST analysis determined 65, 102 and 125 transcripts in human, pig and cattle respectively with at least 10 fold over-expression in the adipose tissue. Based on our comparative functional genomic analysis, seven genes were more abundant and common in investigated mammalian adipose tissues promising a conserved novel gene network in mammalian lipid metabolism. The candidate genes including fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN), Stearoyl-CoA desaturase (SCD) and Lipoprotein lipase (LPL) were selected for further gene expression investigation within two sheep breeds. The real time PCR results showed that among the genes tested, FABP4 was expressed at higher levels than the others. The expression of FABP4 was significantly higher in the fat-tail of Lori-Bakhtiari than in the fat-tail and visceral adipose tissues of Zel (P<0.05). The findings suggest that the FABP4 gene expression in the fat-tail is an important index of fat deposition.

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We previously reported prevention of urolithiasis and associated rat urinary bladder tumors by urine acidification (via diet acidification) in male rats treated with the dual peroxisome proliferator-activated receptor (PPAR)α/γ agonist muraglitazar. Because urine acidification could potentially alter PPAR signaling and/or cellular proliferation in urothelium, we evaluated urothelial cell PPARα, PPARδ, PPARγ, and epidermal growth factor receptor (EGFR) expression, PPAR signaling, and urothelial cell proliferation in rats fed either a normal or an acidified diet for 5, 18, or 33 days. A subset of rats in the 18-day study also received 63 mg/kg of the PPARγ agonist pioglitazone daily for the final 3 days to directly assess the effects of diet acidification on responsiveness to PPARγ agonism. Urothelial cell PPARα and γ expression and signaling were evaluated in the 18- and 33-day studies by immunohistochemical assessment of PPAR protein (33-day study only) and quantitative real-time polymerase chain reaction (qRT-PCR) measurement of PPAR-regulated gene expression. In the 5-day study, EGFR expression and phosphorylation status were evaluated by immunohistochemical staining and egfr and akt2 mRNA levels were assessed by qRT-PCR. Diet acidification did not alter PPARα, δ, or γ mRNA or protein expression, PPARα- or γ-regulated gene expression, total or phosphorylated EGFR protein, egfr or akt2 gene expression, or proliferation in urothelium. Moreover, diet acidification had no effect on pioglitazone-induced changes in urothelial PPARγ-regulated gene expression. These results support the contention that urine acidification does not prevent PPARγ agonist-induced bladder tumors by altering PPARα, γ, or EGFR expression or PPAR signaling in rat bladder urothelium.

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Prostate metabolism is unique, characterised by cholesterol accumulation and reduced respiration. Are these related? We modulated cholesterol levels and despite changes in mitochondrial cholesterol content, we saw no effects on lactate production or respiration. Instead, these features may be related via sterol regulatory element-binding protein 2 (SREBP-2), the master transcriptional regulator of cholesterol synthesis. SREBP-2 diverts acetyl-CoA into cholesterol synthesis and may thus reduce respiration. We examined LNCaP cells overexpressing the SREBP-2 regulator, Scap: although having higher SREBP-2 activity, these cells displayed higher respiration. This striking observation warrants further investigation. Given that SREBP-2 and Scap are regulated by factors driving prostate growth, exploring this observation further could shed light on prostate carcinogenesis.

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Our previous proteomics study revealed that long chain acyl CoA synthetase 1 (ACSL1) and gelsolin (GSN) are oppositely regulated in white adipose tissue of diet-induced obese rats. To firmly establish these proteins as mediators of adipogenic and/or lipogenic events, we efficiently knocked down the Acsl1 and Gsn genes using siRNA in 3T3-L1 adipocyte cells. Expectedly, Acsl1 knockdown stimulated expression of lipogenic genes. Interestingly, Gsn knockdown suppressed expression of lipogenic genes but strikingly increased that of Tnfα and Il6, which may have connections with lipolytic capacity of these genes. Conclusively, we provide clear evidence that ACSL1 and GSN are potential target proteins in the context of obesity.

▼ The aim of the present study was to evaluate the genetic variation of milk fat CLA, together with the variation of CLA precursors, including the individual PUFA that are potentially involved in the metabolic pathway of CLA. The allelic substitution effect, upon these FA, was estimated for a number of polymorphisms, that are located within 9 candidate genes known to influence milk FA partitioning. Sheep milk was chosen because it contains higher total fat percentage, and higher CLA levels per gram fat, than milk of other ruminants. Milk FAME analyses were performed on individual milk samples of sheep from three different breeds. Our results reflect the association of three genes (alpha-1-antichymotrypsin; diacylglycerol O-acyltransferase homolog-2; and zona pellucida glycoprotein-2) with the values of both linolenic FA and CLA; of three more genes (insulin-like growth factor I; lecithin-cholesterol acyltransferase and propionyl coenzyme A carboxylase, beta polypeptide) with the variability of linolenic FA; and of one gene, the fatty acid synthetase with the variability of CLA and stearic FA. The current study might contribute to the understanding of the metabolic pathway of PUFA in sheep milk, providing suggestions to modify milk fat composition through selection.

▼ Benzylamine is found in Moringa oleifera, a plant used to treat diabetes in traditional medicine. In mammals, benzylamine is metabolized by semicarbazide-sensitive amine oxidase (SSAO) to benzaldehyde and hydrogen peroxide. This latter product has insulin-mimicking action, and is involved in the effects of benzylamine on human adipocytes: stimulation of glucose transport and inhibition of lipolysis. This study examined whether chronic, oral administration of benzylamine could improve glucose tolerance and the circulating lipid profile without increasing oxidative stress in overweight and pre-diabetic mice. The benzylamine diffusion across the intestine was verified using everted gut sacs. Then, glucose handling and metabolic markers were measured in mice rendered insulin-resistant when fed a high-fat diet (HFD) and receiving or not benzylamine in their drinking water (3600μmol/(kgday)) for 17 weeks. HFD-benzylamine mice showed lower body weight gain, fasting blood glucose, total plasma cholesterol and hyperglycaemic response to glucose load when compared to HFD control. In adipocytes, insulin-induced activation of glucose transport and inhibition of lipolysis remained unchanged. In aorta, benzylamine treatment partially restored the nitrite levels that were reduced by HFD. In liver, lipid peroxidation markers were reduced. Resistin and uric acid, surrogate plasma markers of metabolic syndrome, were decreased. In spite of the putative deleterious nature of the hydrogen peroxide generated during amine oxidation, and in agreement with its in vitro insulin-like actions found on adipocytes, the SSAO-substrate benzylamine could be considered as a potential oral agent to treat metabolic syndrome.

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Recent studies have demonstrated that FoxO1 modulates the expression of SREBP-1c, but the exact mechanism remains unknown. Our results demonstrate that FoxO1 suppresses the SREBP-1c promoter transcriptional activity in HepG2 cells. This repression was independent of FoxO1 binding to the SREBP-1c promoter, but LXR responsive elements (LXREs) were crucial to this phenomenon. Moreover, FoxO1 also strongly inhibited the LXRα-mediated elevated transcription by SREBP-1c promoter. Electrophoretic mobility shift assay and chromatin immuno-precipitation further suggested the ability of FoxO1 to inhibit LXRα binding with the LXRE in the SREBP-c promoter. FoxO1-mediated suppression of SREBP-1c promoter activity could be partially alleviated by insulin.

▼ Although the safety of gold nanoparticle (AuNP) use is of growing concern, most toxicity studies of AuNPs had focused on their chemical characteristics, including their physical dimensions, surface chemistry, and shape. The present study examined the susceptibility of rodents with healthy or damaged livers to AuNP-induced hepatotoxicity. To induce a model of liver injury, mice were fed a methionine- and choline-deficient (MCD) diet for 4 weeks. Sizes and biodistribution of 15-nm PEGylated AuNPs were analyzed by transmission electron microscopy. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated with an automatic chemical analyzer, and liver sections were subjected to pathological examination. Activities of antioxidant enzymes were determined by biochemical assay. Lateral tail vein injection of MCD diet-fed mice with 5mgkg^-^1 AuNPs significantly elevated the serum ALT and AST levels compared to MCD diet-fed mice injected with mPEG (methylpolyethylene glycol). Similarly, severe hepatic cell damage, acute inflammation, and increased apoptosis and reactive oxygen species (ROS) production were observed in the livers of AuNP-injected mice on the MCD diet; these liver injuries were attenuated in mice fed a normal chow diet. The results suggest that AuNPs display toxicity in a stressed liver environment by stimulating the inflammatory response and accelerating stress-induced apoptosis. These conclusions may point to the importance of considering health conditions, including liver damage, in medical applications of AuNPs.

▼ Glycogen Storage Disease type Ia (GSD-Ia) in humans frequently causes delayed bone maturation, decrease in final adult height, and decreased growth velocity. This study evaluates the pathogenesis of growth failure and the effect of gene therapy on growth in GSD-Ia affected dogs and mice. Here we found that homozygous G6pase (-/-) mice with GSD-Ia have normal growth hormone (GH) levels in response to hypoglycemia, decreased insulin-like growth factor (IGF) 1 levels, and attenuated weight gain following administration of GH. Expression of hepatic GH receptor and IGF 1 mRNAs and hepatic STAT5 (phospho Y694) protein levels are reduced prior to and after GH administration, indicating GH resistance. However, restoration of G6Pase expression in the liver by treatment with adeno-associated virus 8 pseudotyped vector expressing G6Pase (AAV2/8-G6Pase) corrected body weight, but failed to normalize plasma IGF 1 in G6pase (-/-) mice. Untreated G6pase (-/-) mice also demonstrated severe delay of growth plate ossification at 12days of age; those treated with AAV2/8-G6Pase at 14days of age demonstrated skeletal dysplasia and limb shortening when analyzed radiographically at 6months of age, in spite of apparent metabolic correction. Moreover, gene therapy with AAV2/9-G6Pase only partially corrected growth in GSD-Ia affected dogs as detected by weight and bone measurements and serum IGF 1 concentrations were persistently low in treated dogs. We also found that heterozygous GSD-Ia carrier dogs had decreased serum IGF 1, adult body weights and bone dimensions compared to wild-type littermates. In sum, these findings suggest that growth failure in GSD-Ia results, at least in part, from hepatic GH resistance. In addition, gene therapy improved growth in addition to promoting long-term survival in dogs and mice with GSD-Ia.

▼ Background/Aims

Metabolic dysregulation is one of the risk factors for the development of hepatocellular carcinoma (HCC). We investigated the activated metabolic pathway in HCC to identify its role in HCC growth and mortality.

Methods

Gene expression profiles of HCC tissues and non-cancerous liver tissues were obtained by serial analysis of gene expression. Pathway analysis was performed to characterize the metabolic pathway activated in HCC. Suppression of the activated pathway by RNA interference was used to evaluate its role in HCC in vitro. Relation of the pathway activation and prognosis was statistically examined.

Results

A total of 289 transcripts were up- or down-regulated in HCC compared with non-cancerous liver (P<0.005). Pathway analysis revealed that the lipogenic pathway regulated by sterol regulatory element binding factor 1 (SREBF1) was activated in HCC, which was validated by real-time RT-PCR. Suppression of SREBF1 induced growth arrest and apoptosis whereas overexpression of SREBF1 enhanced cell proliferation in human HCC cell lines. SREBF1 protein expression was evaluated in 54 HCC samples by immunohistochemistry, and Kaplan–Meier survival analysis indicated that SREBF1-high HCC correlated with high mortality.

Conclusions

The lipogenic pathway is activated in a subset of HCC and contributes to cell proliferation and prognosis.

▼ Contradictory results have been published regarding the influence of progesterone on lipids metabolism in adipose tissue. The aim of the present work was to elucidate whether progesterone administration in the setting of an experimental model influences lipogenic enzyme genes expression, body and adipose tissue mass. The results presented here indicate that the elevated blood progesterone concentration was associated with significant increase in lipogenic enzyme genes expression in inguinal adipose tissue of females. The rise in the expression of lipogenic enzyme genes was associated with an increase in sterol regulatory element binding transcription factor 1 (Srebf1) and S14 genes expression. Mifepristone, a specific antagonist of progesterone receptor, abolished progesterone's effect on body mass, inguinal fat mass, and lipogenic enzyme genes expression in inguinal adipose tissue. No significant changes were found in the expression of lipogenic enzyme genes, Srebf1 and S14 genes in perirenal white adipose tissue of females. The elevated blood progesterone concentration was associated with the increase in body and inguinal white adipose tissue mass of females. In males, elevated blood progesterone concentration had no effect on the lipogenic enzyme genes expression and on body and fat mass. In conclusion, we demonstrate that a chronic increase in serum progesterone concentration in females was associated with up-regulation of lipogenic enzyme genes expression in inguinal adipose tissue. Up-regulation of Srebf1 and S14 genes expression following progesterone administration suggests that products of these genes might be involved in the regulation of lipogenic enzyme genes expression by progesterone. The stimulatory effect of progesterone on lipogenic enzyme genes expression in inguinal adipose tissue seems to be specific as it was reversed by specific antagonist of progesterone receptor.

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To investigate tissue fatty acid distribution in relation to the incidence of colorectal cancer prognosis, adjacent normal tissue and cancerous tissue from 35 samples of clinically incident colorectal cancer were obtained. Fatty acids were measured in the colorectal mucosa phospholipid fraction by gas chromatography mass spectrometry. Palmitoleic acid and oleic acid were significantly lower in colorectal cancerous tissue, ranging from 20% to 50% less than the adjacent normal tissue. The omega-6 (n-6) fatty acid family members (20:2, 20:3, 20:4 and 22:4) were higher by 1–3 fold in cancerous colorectal tissue. Contrary with the high level of n-6 fatty acids, about a 37% to 87% reduction in EPA and DHA was observed in colorectal cancerous tissue. A higher level of linoleic acid and arachidonic acid was detected in the C cancer stage than in the B cancer stage (p<0.05), but a lower level of oleic acid and docosahexenoic acid was detected in the C cancer stage (p<0.05). The fatty acid distribution of colorectal tissue is strongly linked to the incidence of colorectal cancer. This study also provides scientific basis for identifying novel biomarkers for the diagnosis and treatment of cancer.

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A medium-chain-triglyceride (MCT)-based diet is mainstay of treatment in very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), a long-chain fatty acid β-oxidation defect. Beneficial effects have been reported with an MCT-bolus prior to exercise. Little is known about the impact of a long-term MCT diet on hepatic lipid metabolism. Here we investigate the effects of MCT-supplementation on liver and blood lipids in the murine model of VLCADD.

Wild-type (WT) and VLCAD-knock-out (KO) mice were fed (1) a long-chain triglyceride (LCT)-diet over 5weeks, (2) an MCT diet over 5weeks and (3) an LCT diet plus MCT-bolus. Blood and liver lipid content were determined. Expression of genes regulating lipogenesis was analyzed by RT-PCR.

Under the LCT diet, VLCAD-KO mice accumulated significantly higher blood cholesterol concentrations compared to WT mice. The MCT-diet induced severe hepatic steatosis, significantly higher serum free fatty acids and impaired hepatic lipid mobilization in VLCAD-KO mice. Expression at mRNA level of hepatic lipogenic genes was up-regulated.

The long-term MCT diet stimulates lipogenesis and impairs hepatic lipid metabolism in VLCAD-KO mice. These results suggest a critical reconsideration of a long-term MCT-modified diet in human VLCADD. In contrast, MCT in situations of increased energy demand appears to be a safer treatment alternative.

▼ Objectives

The Liver X receptors (LXR) alpha and beta and their target genes such as the ATP-binding cassette (ABC) transporters have been shown to be crucially involved in the regulation of cellular cholesterol homeostasis. The aim of this study was to characterize the role of LXR alpha/beta in the human placenta under normal physiological circumstances and in preeclampsia.

Study design

We investigated the expression pattern of the LXRs and their target genes in the human placenta during normal pregnancy and in preeclampsia. Placental explants and cell lines were studied under different oxygen levels and pharmacological LXR agonists.

Main outcome measures

Gene expressions (Taqman PCR) and protein levels (Western Blot) were combined with immunohistochemistry to analyze the expression of LXR and its target genes.

Results

In the human placenta, LXRA and LXRB expression increased during normal pregnancy. This was paralleled by the expression of their prototypical target genes, e.g., the cholesterol transporter ABCA1. Interestingly, early-onset preeclamptic placentae revealed a significant upregulation of ABCA1. Culture of JAr trophoblast cells and human first trimester placental explants under low oxygen lead to increased expression of LXRA and ABCA1 which was further enhanced by the LXR agonist T0901317.

Conclusions

LXRA together with ABCA1 are specifically expressed in the human placenta and can be regulated by hypoxia. Deregulation of this system in early preeclampsia might be the result of placental hypoxia and hence might have consequences for maternal-fetal cholesterol transport.

▼ Experimental studies have demonstrated that dietary macronutrient distribution plays an important role in insulin regulation, a risk factor associated to obesity, diabetes and other metabolic disorders. To assess whether the macronutrient composition of the diet could be related to obesity onset by affecting the epigenetic regulation of gene expression, we investigated in rats the metabolic effects of two pair-fed isocaloric diets: control (rich in carbohydrates) and high fat diet (rich in fat; HFD). Compared to controls, HFD induced higher weight gain and adiposity and impaired glucose tolerance, which was accompanied by a slight increase in adiponectin levels and liver steatosis. Epididymal adipose tissue expression of the fatty acid synthase (FASN) gene and NADH dehydrogenase (ubiquinone) 1β-subcomplex 6 (NDUFB6) were significantly reduced in HFD group. These variations in mRNA levels were accompanied by changes in the methylation patterns of several CpG islands located in the promoter region of these genes. However, no correlations were found between gene expression and the methylation status. These results suggest that high fat intake produces overweighted rats independently of total energy intake. These diets could also induce some epigenetic changes in the promoters of key genes that could influence gene expression and may be behind metabolic alterations.

▼ Background: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common long-chain fatty acid oxidation defect presenting with heterogeneous clinical phenotypes. Dietary fat plays a crucial role in disease pathogenesis and fat restriction is a common treatment measure. We here investigate the hepatic and muscular effects of a fat-enriched and a fat-restricted diet. Methods: VLCAD knock-out (KO) and wild-type (WT) mice are subjected to a fat-rich (10.6%), a fat-reduced (2.6%) or a regular mouse diet (5.1%) for 5weeks. Analyses are performed at rest and after one hour exercise on a treadmill. Acylcarnitines in muscle as well as lipid and glycogen content in muscle and liver are quantified. Expression of genes involved in lipogenesis is measured by Real-Time-PCR. Results: At rest, VLCAD KO mice develop no clinical phenotype with all three diets, but importantly VLCAD KO mice cannot perform one hour exercise as compared to WT, this is especially apparent in mice with a fat-reduced diet. Moreover, changes in dietary fat content induce a significant increase in muscular long-chain acylcarnitines and hepatic lipid content in VLCAD KO mice after exercise. A fat-reduced diet up-regulates hepatic lipogenesis at rest. At the same time, muscular glycogen is significantly lower than in WT. Conclusions: We here demonstrate that a fat-reduced and carbohydrate-enriched diet does not prevent the myopathic phenotype in VLCAD KO mice. An increase in dietary fat is safe at rest with respect to the muscle but results in a significant muscular acylcarnitine increase after exercise.

▼ Gemcitabine (2'-deoxy-2', 2'-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27^K^i^p^1 protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27^K^i^p^1 protein degradation. The induction of UBE2M and reduction of p27^K^i^p^1 by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27^K^i^p^1. Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27^K^i^p^1. Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27^K^i^p^1 expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27^K^i^p^1 in drug resistance.

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Hepatitis C virus (HCV) is often associated with steatosis, cirrhosis and hepatocellular carcinoma (HCC). Statins (HMG-CoAR inhibitors) have been shown to exert an antiviral effect in vitro, principally on replicon harboring cells, but the effect of their use alone in vivo remains controversial. In clinical trials, when used in combination with the standards of care (SOC), they led to an increased proportion of sustained virological responder (SVR). Here we investigated the implication of SKI-1/S1P, a master lipogenic pathways regulator upstream of HMG-CoAR, on different steps of HCV life cycle. We compared the HCV antiviral effect of the most potent SKI-1/S1P small molecule inhibitor (PF-429242) with a set of two statins on different steps of the viral life cycle, and showed that SKI-1/S1P inhibitor blocked HCVcc (strain JFH-1) RNA replication (EC₅₀= 5.8μM) more efficiently than statins. Moreover, we showed that PF-429242 could reduce lipid droplets accumulation in Huh7 cells. Interestingly, PF-429242 dramatically reduced infectious particles production (EC₉₀= 4.8μM). Such inhibition could not be achieved with statins. SKI-1/S1P activity is thus essential for viral production and its inhibition should be considered for antiviral drug development.