SREBP-1 Gene Expression Research Articles

Three-Dimensional Bioprinting of Tarsal Plates with Adipose-Derived Mesenchymal Stem Cells: Evaluation of Meibomian Gland Reconstruction in a Rat Model.

Background: The aim of this study was to develop 3D-bioprinted scaffolds embedded with human adipose-derived stem cells (hADSCs) to reconstruct the tarsal plate in a rat model. Methods: Scaffolds were printed using a 3D bioprinter with a bioink composed of atelocollagen and alginate. hADSCs (5 × 105 cells/mL) were embedded within the bioink. A total of 30 male Sprague Dawley (SD) rats (300 g) were divided into three groups: group 1 (normal control, n = 10), group 2 (3D-bioprinted scaffolds, n = 10), and group 3 (3D-bioprinted scaffolds with hADSCs, n = 10). Four weeks after surgery, a histopathological analysis was performed using hematoxylin and eosin (H&E) staining, Masson's trichrome (MT) staining, and immunofluorescence staining. Gene expression of SREBP-1, PPAR-γ, FADS-2, and FAS was assessed via real-time polymerase chain reaction (PCR). Results: No abnormalities were observed in the operated eyelids of any of the 30 rats. The histopathological analysis revealed lipid-secreting cells resembling meibocytes in both group 2 and group 3, with more pronounced meibocyte-like cells in group 3. Immunofluorescence staining for phalloidin expression showed a significant increase in group 3. Additionally, the RNA expression of SREBP-1, PPAR-γ, FADS-2, and FAS, all related to lipid metabolism, was elevated in group 3. Conclusions: The 3D-printed scaffolds combined with hADSCs were effective for tarsal plate reconstruction, with the hADSCs notably contributing to the generation of cells associated with lipid metabolism.

Bta-miR-365-3p-targeted FK506-binding protein 5 participates in the AMPK/mTOR signaling pathway in the regulation of preadipocyte differentiation in cattle.

MicroRNAs (miRNAs) are endogenous non-coding RNAs that can play a role in the post-transcriptional regulation of mammalian preadipocyte differentiation. However, the precise functional mechanism of its regulation of fat metabolism is not fully understood. We identified bta-miR-365-3p, which specifically targets the 3' untranslated region (3'UTR) of the FK506-binding protein 5 (FKBP5), and verified its mechanisms for regulating expression and involvement in adipogenesis. In this study, we found that the overexpression of bta-miR-365-3p significantly decreased the lipid accumulation and triglyceride content in the adipocytes. Compared to inhibiting bta-miR-36 5-3p group, overexpression of bta-miR-365-3p can inhibit the expression of adipocyte differentiation-related genes C/EBPα and PPARγ. The dualluciferase reporter system further validated the targeting relationship between bta-miR-365-3p and FKBP5. FKBP5 mRNA and protein expression were detected by quantitative real-time polymerase chain reaction and Western blot. Overexpression of bta-miR-365-3p significantly down-regulated FKBP5 expression, while inhibition of bta-miR-365-3p showed the opposite, indicating that bta-miR-365-3p negatively regulates FKBP5. Adenosine 5'-monophosphate (AMP)-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway is closely related to the regulation of cell growth and is involved in the development of bovine adipocytes. In this study, overexpression of bta-miR-365-3p significantly inhibited mRNA and protein expression of AMPK, mTOR, and SREBP1 genes, while the inhibition of bta-miR-365-3p expression was contrary to these results. Overexpression of FKBP5 significantly upregulated AMPK, mTOR, and SREBP1 gene expression, while inhibition of FKBP5 expression was contrary to the above experimental results. In conclusion, these results indicate that bta-miR-365-3p may be involved in the AMPK/mTOR signaling pathway in regulating Yanbian yellow cattle preadipocytes differentiation by targeting the FKBP5 gene.

The potential efficacy of Artemisia anuua L. extract nanoparticles in mitigating obesity-related-metabolic complications in hypercaloric diet-fed rats

ABSTRACT Obesity-related health problems are worsening in various societies worldwide. Artemisia anuua L. (AA) has distinctive health benefits, including anti-inflammatory, antioxidant, and insulin-sensitizing properties. Nanoformulation of herbal extracts is a new approach to enhance the bioavailability of natural products. This study aimed to evaluate the efficacy of AA extract, either the conventional extract (AAE) or its nano-form (AAEN), in modulating obesity-related metabolic complications in hypercaloric (high-fat/high-sucrose) diet-induced obese rats. Forty-eight rats were divided into two groups fed either a basal or hypercaloric diet for twelve weeks. Starting from the eighth week, each main group was subdivided into three subgroups and treated orally with either saline, AAE, or AAEN until the end of the experiment. Both AAE and AAEN exerted antiadipogenic effects. They reduced body weight and epididymal fat, mitigated dyslipidemia, improved glucose utilization and insulin resistance, reduced hyperleptinemia, increased serum adiponectin, and decreased oxidative stress and inflammatory markers. These results were mediated by regulating SIRT-1, PGC-1α, SREBP-1, PPAR-γ, FAS, LPL, IRS 1, and GLUT4 gene expression, suggesting an effect of AA on de novo lipogenesis and glucose utilization. The AAEN produced more expressive ameliorating effects than the conventional extract. AAE administration ameliorated the obesity-associated metabolic complications induced by feeding a hypercaloric diet, implying that this extract may be used as a complementary treatment option for obesity and its metabolic consequences. The antiadipogenic effectiveness of AAE was enhanced by its nanoformulation, suggesting that the nanoparticle system can be applied to boost other herbal medicine effectiveness.

Application of sunflower meal in diets of on-growing grass carp (Ctenopharyngodon idellus) and evaluation of enzymatic hydrolysis

An 8-week trial was performed to investigate the effects of replacing soybean meal (SBM) by sunflower meal (SM) or enzymolysis sunflower meal (ESM) on the growth, lipid metabolism, and intestinal health of Ctenopharyngodon idellus. Compared with SM, the small peptides and amino acids contents in ESM increased, while the crude fiber decreased. Seven isonitrogenous and isolipid diets (SM0, SM25, SM50, SM75, SM100, ESM75, and ESM100) were prepared for SM to replace 0, 25, 50, 75, 100% SBM and ESM to replace 75 and 100% SBM, respectively. The results showed that with an increasing proportion of SM instead of SBM, weight gain rate (WGR) of grass carp showed a decreasing trend, while feed conversion ratio (FCR) showed a rising trend, and significantly changed in the SM75 and SM100 groups. WGR in the ESM75 group was significantly increased compared with the SM75 group, and there was no statistical difference with the SM0 group. Replacing 75% and 100% SBM with ESM was beneficial to lipid metabolism, which was mainly manifested in decreased serum low-density lipoprotein, triglyceride, and total cholesterol contents, and significantly down-regulated acetyl-CoA carboxylase (acc), fatty acid synthase (fas) and sterol regulatory element binding protein 1 (srebp1) gene expression in liver. Replacing 75% and 100% SBM with SM resulted in intestinal oxidative damage (reactive oxygen species and malondialdehyde contents increased; superoxide dismutase, glutathione reductase, and glutathione peroxidase activities decreased) and disrupted intestinal physical and immune barrier functions (claudin15, myosin light chain kinase (mlck), interleukin-1β, 6, 12β (il-1β, il-6, il-12β), tumor necrosis factor-α (tnf-α), and nuclear factor-κB (nf-κb) gene expression up-regulated; zonula occluden 1, 2 (zo1, zo2), transforming growth factor β1 (tgf-β1), and il-10 gene expression down-regulated), while substitution of 75% and 100% SBM by ESM alleviated oxidative damage and improved intestinal immune and physical barrier functions. In addition, the α-diversity index and the abundances of Erysipelatoclostridium and Bacteroides in the ESM75 group were significantly increased compared with the SM0 and SM75 groups. In conclusion, the replacement proportion of SBM with SM should not exceed 50%, otherwise it will lead to reduced growth, lipid deposition, intestinal health damage and intestinal microbiota disorder. However, the replacement of 75% SBM with ESM has no negative effect on grass carp, and it improved lipid metabolism and intestinal health compared with substitution of SBM with SM.

Kombucha tea improves glucose tolerance and reduces hepatic steatosis in obese mice

Nonalcoholic fatty liver disease (NAFLD), often associated with obesity, is becoming one of the most common liver diseases worldwide. It is estimated to affect one billion individuals and may be present in approximately 25% of the population globally. NAFLD is viewed as a hepatic manifestation of metabolic syndrome, with humans and animal models presenting dyslipidemia, hypertension, and diabetes. The gut-liver axis has been considered the main pathogenesis branch for NAFLD development. Considering that foods or beverages could modulate the gastrointestinal tract, immune system, energy homeostasis regulation, and even the gut-liver axis, we conducted an exploratory study to analyze the effects of kombucha probiotic on hepatic steatosis, glucose tolerance, and hepatic enzymes involved in carbohydrate and fat metabolism using a pre-clinical model. The diet-induced obese mice presented glucose intolerance, hyperinsulinemia, hepatic steatosis, increased collagen fiber deposition in liver vascular spaces, and upregulated TNF-alpha and SREBP-1 gene expression. Mice receiving the kombucha supplement displayed improved glucose tolerance, reduced hyperinsulinemia, decreased citrate synthase and phosphofructokinase-1 enzyme activities, downregulated G-protein-coupled bile acid receptor, also known as TGR5, and farnesol X receptor gene expression, and attenuated steatosis and hepatic collagen fiber deposition. The improvement in glucose tolerance was accompanied by the recovery of acute insulin-induced liver AKT serine phosphorylation. Thus, it is possible to conclude that this probiotic drink has a beneficial effect in reducing the metabolic alterations associated with diet-induced obesity. This probiotic beverage deserves an extension of studies to confirm or refute its potentially beneficial effects.

Exploring the Possible Link between the Gut Microbiome and Fat Deposition in Pigs.

Excessive lipid accumulation and high oxidative stress have become a serious health and economic problem in the pig industry. Fatness characteristics are crucial in pig production since they are closely related to meat quality. The gut microbiome is well acknowledged as a key element in fat deposition. But the link between gut microbiota and fat accumulation in pigs remains elusive. To examine whether there is a link between pigs' gut microbiome, lipogenic properties, and oxidative stress, we selected 5 high-fat pigs and 5 low-fat pigs from 60 250-day-old Jinhua pigs in the present study and collected the colon content, serum sample, and liver and abdominal fat segments from each pig for metagenomic analysis, the oxidative stress assay, and RT-qPCR analysis, respectively. The backfat thickness and fat content of the longissimus dorsi muscle were considerably higher in the high-fat pigs than in the low-fat pigs (P < 0.05). An obvious difference in GSH-Px and MDA in the serum between the high- and low-fat pigs was observed. After RT-qPCR analysis, we found the gene expression of ACC1 and SREBP1 in the liver and FAS, PPARγ, and LPL in the abdominal fat were significantly higher in high-fat pigs than in low-fat pigs (P < 0.05). Additionally, metagenomic sequencing revealed that high-fat pigs had a higher abundance of Archaeal species with methanogenesis functions, leading to more-efficient fat deposition, while low-fat pigs had higher abundances of butyrate-producing bacteria species that improved the formation of SCFAs, especially butyrate, thus alleviating fat deposition in pigs. Furthermore, a total of 17 CAZyme families were identified to give significant enrichments in different fat phenotypes of pigs. This study would provide a detailed understanding of how the gut microbiome influences fat deposition in pigs, as well as a hint for improving growth performance and fatness traits by manipulating the gut microbiome.

Improving dairy performance through molecular characterization of SREBP-1 gene in Sarda sheep breed

This research has two aims: (i) to characterize the coding sequence of the SREBP-1 gene in dairy sheep in order to investigate possible relationships between single nucleotide polymorphisms (SNPs) and milk traits; and (ii) to investigate possible relationship between SREBP-1 gene expression and nucleotide variation. Four hundred adult and multiparous lactating Sarda breed ewes were selected from two farms. Milk samples were collected from Day 30 to Day 150 of lactation to determine the mean yield, somatic cell count, lactose, fat, and protein content of the milk. RNA was extracted from the milk samples, after which the SREBP-1 gene coding regions were amplified and sequenced to scan mutations. Whilst eight SNPs were identified, none had statistically significant association with the analysed milk traits. Moreover, the identified expression patterns were not affected by the SNP or combined genotypes. High SREBP-1 gene expression levels were found to be correlated with high milk fat content (P < 0.01), indicating the crucial role of this gene in the milk fat synthesis. In conclusion, the polymorphisms found within SREBP-1 gene exhibited no significant associations with milk traits or with individual SREBP-1 mRNA expression patterns. The findings thus suggest that this small genetic variability may derive from the selection carried out in Sarda breed to improve milk yield.

Effect of a genetic polymorphism in SREBP1 on fatty acid composition and related gene expression in subcutaneous fat tissue of beef cattle breeds.

Sterol regulatory element-binding factor 1 (SREBP1) plays an important role in the lipogenesis which affects fatty acid (FA) composition in backfat and consequently influences beef nutritional quality. This study analyzed the association of 84bp-indel, both short (S) and long (L) alleles in intron 5 of SREBP1, with FA composition and gene expression of SREBP1 in backfat of northern Spanish beef breeds (Pirenaica, Salers and Holstein-Friesian). Phylogenetic analysis suggests that 84bp-indel of ruminants is a highly conserved region compared with those in the full-length sequence of intron 5 or mRNA of SREBP1 among species. Overall, higher content of polyunsaturated FAs was observed in SL genotype compared to LL genotype of 84bp-Indel (p<.05). In particular, in Pirenaica, SL genotype was associated with a higher content of stearic (18:0), α-linolenic (18:3n-3) acid, and total n-3 content (p<.05). However, the gene expression of SREBP1 did not differ among genotypes of 84bp-Indel (p>.05).

Improvement of Hypertriglyceridemia by Roasted Nelumbinis folium in High Fat/High Cholesterol Diet Rat Model.

Hypertriglyceridemia is a condition characterized by high triglyceride levels and is a major risk factor for the development of cardiovascular diseases. The present study was designed to investigate the inhibitory effect of roasted Nelumbinis folium (RN), which is a medicinal substance produced by heating lotus leaves, on lipid metabolism in high fat/cholesterol (HFC) diet-induced hypertriglyceridemia. Except for those in the control group, Sprague–Dawley rats were fed an HFC diet for four weeks to induce hypertriglyceridemia. During the next nine weeks, the control, regular diet; HFC, HFC diet, FLU, fluvastatin (3 mg/kg/day); RNL, RN (100 mg/kg/day); RNH, RN (200 mg/kg/day) were orally administered together with the diet, and the experiments were conducted for a total of 13 weeks. The weight of the epididymal adipose tissue, liver, and heart of rats in the HFC diet group significantly increased compared to those in the control group but improved in the RN-treated group. It was also confirmed that vascular function, which is damaged by an HFC diet, was improved after RN treatment. The levels of insulin, glucose, triglycerides, total cholesterol, and low-density lipoprotein increased in the HFC diet group compared to those in the control group, while the administration of RN attenuated these parameters. In addition, the administration of RN significantly reduced the gene expression of both LXR and SREBP-1, which indicated the inhibitory effect of the biosynthesis of triglycerides caused by RN. The results indicated that RN administration resulted in an improvement in the overall lipid metabolism and a decrease in the concentration of triglycerides in the HFC diet-induced rat model of hypertriglyceridemia. Therefore, our findings suggest that the RN can be a candidate material to provide a new direction for treating hypertriglyceridemia.

Niacin inhibits the synthesis of milk fat in BMECs through the GPR109A-mediated downstream signalling pathway

AimsPrevious studies have shown the effect of niacin on dairy cow production, but no study on the role of niacin in milk fat synthesis has been performed. Therefore, the purpose of this study was to examine the effect of niacin on milk fat synthesis and its specific mechanism in BMECs. Main methodsIn this study, 0.5 mM niacin, a GPR109A-inhibiting plasmid, and an AMPK inhibitor were added to BMECs. Milk fat was measured by a triglyceride kit and BODIPY staining. The protein expression of GPR109A, FASN, SREBP1, AMPK, ACC, mTOR and S6K was measured by Western blotting. The gene expression of GPR109A, FASN, and SREBP1 was analysed by RT-PCR. Key findingsOur results showed that 0.5 mM niacin could significantly reduce milk fat synthesis in BMECs and activate the AMPK/ACC signalling pathway by stimulating GPR109A, reducing the protein expression of p-mTOR and p-S6K, and reducing the expression of SREBP1 and FASN in BMECs. SignificanceThe present study clarified the effect of niacin on milk fat synthesis. The results show that niacin inhibits the synthesis of milk fat in BMECs through the downstream signalling pathway mediated by GPR109A. The function of niacin has been expanded, and knowledge of the new mechanism and signalling pathway will help improve the biosynthesis of milk fat.

Effects of Insulin on Milk Fat Synthesis by Regulating the Expressions of SREBP1 and mTOR in Bovine Mammary Epithelial Cells

Due to the complexity of insulin in life activities, the role of insulin in mammalian lactation has not been well explained. To investigate the influence of insulin on milk fat synthesis, bovine mammary epithelial cells (BMECs) were cultured in treatment with insulin. We determined the content of Triglyceride (TG) in cell-free culture medium and found a notable increase in TG secrection. Lipid droplet staining study showed a consistent result. We also used real-time quantitative PCR and western blotting to detect the expression of signaling molecules related to milk fat synthesis. We found that insulin resulted in an obvious increase of SREBP-1, mTOR and lipogenic gene expression compared with the blank group. Taken together, our study reveals that insulin plays a significant role in milk fat synthesis.

Effects of sex and fasting/refeeding on hepatic AMPK signaling in chickens (Gallus gallus)

The alpha-1 isoform of chicken AMPK situates on the Z-chromosome, in contrast, the other isoforms in birds and the mammalian AMPKα1 are located on the autosomes. The present study aimed to investigate the role of hepatic AMPK signaling in adaptation to nutritional status and the potential sex-specific response in chickens. Hepatic genes and proteins were compared between the two sexes immediately after hatching. From 20d of age, chicks from each sex received feed treatments: Control was fed ad libitum; Fasted was starved for 24 h; Refed was fed for 4 h after a 24 h fasting. As a result, hepatic AMPKα1 mRNA level in males was significantly higher at both ages compared to females, due to the presence of Z-chromosomes. However, this did not make this kinase “male-bias” as it was eventually compensated at a translational level, which was not reported in previous studies. The protein levels and activation of AMPKα were even lower in newly-hatched male compared to female chicks, accompanied with a higher FAS and SREBP-1 gene expressions. Accordingly, hepatic G6PC2 mRNA levels in males were significantly lower associated with lower plasma glucose levels after hatching. Fasting activated hepatic AMPK, which in turn inhibited gene expression of GS, FAS and SREBP-1, and stimulated the downstream G6PC2 in both sexes. These changes recovered after refeeding. In conclusion, AMPK plays a role in adaptation to nutritional environment for both sexes. The Z-linked AMPK did not exert a sex-specific signaling, due to a “translational compensation” of AMPKα1.

Activation of peroxisome proliferator-activated receptor gamma/small heterodimer partner pathway prevents high fat diet-induced obesity and hepatic steatosis in Sprague–Dawley rats fed soybean meal

Soybeans are a complete nutritional resource and soybean proteins are known to affect lipid metabolism via multiple mechanisms. Soybean meal (SBM) is produced after extraction of soybean oil and in this study, we investigated the ability whether the SBM could prevent high fat diet-induced obesity and understand the underlying mechanisms. Male Sprague–Dawley rats, aged 5 weeks, were randomly divided into three groups (n=8 each) and fed one of three diets for 28 days: Cont (AIN-93G), HFD (high fat diet with 40% of calories derived from fat) and HFD+SBM (HFD with 30% SBM). White adipose tissue weight as well as plasma and hepatic triglycerides were significantly decreased in HFD+SBM rats. Expression of hepatic SREBP-1 and its target genes was significantly decreased in HFD+SBM rats. Meanwhile, expression of SHP gene expression was significantly increased in HFD+SBM, and there was a negative correlation between SHP and SREBP-1 expression. Together these results suggest that hepatic SREBP-1 gene expression is negatively regulated by SHP. Expression of PPARG, the transcriptional factor that regulates SHP expression, was increased in HFD+SBM rats. Furthermore, expression of genes controlled by PPARG/SHP, such as those involved in hepatic gluconeogenesis, was also significantly decreased in HFD+SBM rats. Therefore, in addition to the previous findings of SBM on obesity here we show an additional mechanism which by changing the expression of genes involved in lipid metabolism via the PPARG/SHP pathway in the liver.

Lipodystrophy-like features after total body irradiation among survivors of childhood acute leukemia.

Background/objectiveThe number of long-term survivors of childhood acute leukemia (AL) is substantially growing. These patients are at high risk for metabolic syndrome (MS), especially those who received total body irradiation (TBI). The consequences of children’s irradiation on adipose tissue (AT) development in adulthood are currently unknown. The objective of this study is to assess the impact of TBI on AT of childhood AL survivors.DesignWe compared the morphological and functional characteristics of AT among survivors of childhood AL who developed MS and received (n = 12) or not received (n = 12) TBI.Subjects/methodsBody fat distribution and ectopic fat stores (abdominal visceral and liver fat) were evaluated by DEXA, MRI and 1H-spectroscopy. Functional characteristics of subcutaneous AT were investigated by studying gene expression and pre-adipocyte differentiation in culture.ResultsPatients who have received TBI exhibited a lower BMI (minus 5 kg/m2) and a lower waist circumference (minus 14 cm), especially irradiated women. Despite the lower quantity of intra-abdominal AT, irradiated patient displayed a nearly two-fold greater content of liver fat when compared to non-irradiated patient (17 vs 9%, P = 0.008). These lipodystrophic-like features are supplemented by molecular abnormalities in subcutaneous AT of irradiated patients: decrease of gene expression of SREBP1 (minus 39%, P = 0.01) and CIDEA (minus 36%, P = 0.004) and a clear alteration of pre-adipocyte differentiation.ConclusionsThese results strongly support the direct effect of irradiation on AT, especially in women, leading to specific nonalcoholic fatty liver disease, despite lower BMI. A long-term appropriate follow-up is necessary for these patients.

Effect of potential probiotic Leuconostoc mesenteroides FB111 in prevention of cholesterol absorption by modulating NPC1L1/PPARα/SREBP-2 pathways in epithelial Caco-2 cells.

Mustard kimchi consumption reduces cholesterol levels in rats. To identify lactic acid bacteria (LAB) in kimchi which exert this effect, 20 LAB isolates were evaluated for cholesterol reduction in an in vitro screen. The FB111 strain showed the highest cholesterol-lowering activity and was identified as Leuconostoc mesenteroides. This strain was characterized as a potential probiotic through sequential analyses for resistance to gastrointestinal digestion and bile salts, and adhesion to Caco-2 cells. The Caco-2 cells treated with L. mesenteroides FB111 (6-8 log CFU/mL) showed toxicological effect. The reduction of cholesterol uptake in these cells was inhibited by 48.6% compared to the control and significantly higher than that of the Lactobacillus rhamnosus GG (LGG) strain-treated group after 2-h incubation. The levels of NPC1L1, ABCG5, ABCG8, SREBP-1, SREBP-2, and PPARα gene expression were determined by reverse transcription-quantitative polymerase chain reaction analysis. The L. mesenteroides FB111 and LGG inhibited the mRNA expression of NPC1L1 (P < 0.05), whereas the expression of PPARα was increased. Moreover, the FB111 strain also inhibited the expression of SREBP-2 mRNA. Overall, we found that L. mesenteroides FB111 has efficient cholesterol-lowering effects and might be useful as a probiotic in the food industry.

Establishment of a HEK293 cell line by CRISPR/Cas9-mediated luciferase knock-in to study transcriptional regulation of the human SREBP1 gene.

To establish a HEK293 cell line with a luciferase knock-in reporter controlled by the endogenous SREBP1 promoter for investigating transcriptional regulation of the SREBP1 gene. PCR confirmed the site-specific integration of a single copy of the exogenous luciferase gene into one allele of the genome and a 14bp deletion of the targeted sequence in the other. Luciferase activity was directly correlated with the promoter activity of the endogenous SREBP1 gene in the HEK293-SREBP1-T2A-luciferase-KI cell line cell line. We successfully generated anovel luciferase knock-in reporter system, which will be very useful for studying transcriptional regulation of the SREBP1 gene and for screening drugs or chemical molecules that regulate SREBP1 gene expression.

Abstract 457: Feedback Regulation of 3-Hydroxy-3-methylglutaryl-coenzyme a Reductase in Livers of Mice

Introduction: HMG-CoA reductase is a membrane protein of the endoplasmic reticulum that catalyzes reduction of HMG-CoA to mevalonate, a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, which exert feedback control on HMGCR through multiple mechanisms. These mechanisms ensure constant synthesis of essential nonsterol isoprenoids, while avoiding toxic cholesterol accumulation. One mechanism involves sterol-induced ubiquitination of HMGCR, marking the enzyme for degradation from ER membranes, a process augmented by nonsterol isoprenoids. We examine the contribution of sterol-accelerated ubiquitination/degradation to overall regulation of HMGCR in livers of mice. Methods: Forty mice, including 20 wild-type (WT) and 20 knock-in (Ki) mice expressing ubiquitination-resistant HMGCR, were fed diets containing only chow, or chow supplemented with 0.1, 0.3, or 1% cholesterol. After five days of feeding, livers were harvested for measurements of cholesterol and triglycerides, immunoblot analysis, and qRT-PCR of genes related to cholesterol, nonsterol isoprenoid, and fatty acid synthesis. Results: Normalization of mRNA to protein levels indicates that HMGCR Ki livers contain a more HMGCR protein despite mRNA downregulation. Protein and gene expression of SREBP2 and its target genes, which contribute to cholesterol synthesis, decreased as expected with increased dietary cholesterol. Conversely, protein and gene expression of SREBP1 and its target genes increased, likely due to SREBP1c predominance toward fatty acid synthesis, which prevents cholesterol accumulation. Conclusion: The increase in HMGCR protein relative to mRNA suggests that significant post-transcriptional regulation exists in the form of impaired degradation. Furthermore, these normalized values indicate that accumulation of protein is primarily due to impaired degradation at lower cholesterol levels (chow, 0.1%); however, at high cholesterol levels (0.3, 1%), a greater degree of transcriptional control from sterol-mediated inhibition of SREBP2 regulates HMGCR due to negative feedback. This study demonstrates the role of degradative control on inhibition of HMGCR and may assist in reducing HMGCR accumulation during statin therapy.

Abstract 386: Dietary Oxidized Linoleic Acid Modulates PCSK9 and Associated Genes

Introduction: We have previously shown that oxidized linoleic acid (OxLA) modulate plasma lipids. In this study we investigated the role of dietary OxLA on PCSK-9 and its associated genes. Method: Liver tissue from four groups of normal C57BL6 mice were feed; plain chow, chow supplemented with linoleic acid (9mg), chow supplemented with a low concentration of OxLA (9mg) and chow supplemented with a high concentration of OxLA (18mg) were analyzed for PCSK-9 and related genes. RNA from these livers samples was extracted with Trizol, assessed for purity by agarose gel electrophoresis and quantified using fluorometry. cDNA was synthesized from total extracted RNA and subsequently used for qRT-PCR analysis. Gene expressions of PCSK-9, SREBP-1, HNF-1α and PPARα were measured and normalized to GAPDH . Total, free and cholesterol ester levels in the liver were analyzed spectrophotometrically. Experiments were performed in triplicates and the difference between means was determined using a student’s T-test with a p value of &lt;0.05 accepted as statistically significant. Results: PCSK-9 gene expression was markedly elevated in the mice fed high concentration OxLA, SREBP-1 gene levels in these of mice was also significantly elevated in comparison to the other 3 groups which had relatively the same levels of SREBP-1 gene expression. Conversely HNF-1α gene expression levels were elevated in all groups except the mice on low concentration OxLA. PPARα gene levels were significantly down regulated in the mouse on high concentration OxLA. Plasma total cholesterol levels were significantly elevated in both groups on OxLA. Conclusion: Elevated cholesterol levels in the livers of mice fed high concentration OxLA was accompanied with increased PCSK-9 expression. These results show that OxLA modulate PCSK-9 and its associated genes expression and promote accumulation of liver cholesterol.

Food-drug interaction: Anabolic steroids aggravate hepatic lipotoxicity and nonalcoholic fatty liver disease induced by trans fatty acids

Remains unknown if dietary lipids and anabolic steroids (AS) can interact to modify energy metabolism, hepatic structure and function. We investigated the impact of AS on gene expression, lipid profile, redox status and the development of nonalcoholic fatty liver disease (NAFLD) in mice treated with a diet rich in trans fatty acids. Seventy-two C57BL/6 mice were equally randomized into six groups and treated with a standard diet (SD) or high-fat diet (HFD) alone or combined with testosterone cypionate (10 or 20 mg/kg) for 12 weeks. When combined with a HFD, AS reduced plasma HDL cholesterol levels. It also upregulated SREBP-1, PPARα, SCD-1 and ACOX1 gene expression; plasma and hepatic triglyceride levels; oxidative stress; circulating hepatic transaminase levels and NAFLD severity. Our finding indicated that the activity of antioxidant enzymes such as catalase, glutathione-s-transferase and superoxide dismutase was attenuated by HFD, an effect whose implications for AS-induced hepatotoxicity requires further investigation. Increased lipid, protein and DNA oxidative damage as well as worsening NAFLD in response to the interaction of HFD and AS were also potentially associated with the ability of AS to amplify the activation of regulatory lipid metabolism genes that are also involved in the control of cellular redox balance.

Feedback Regulation of HMG‐CoA Reductase in Livers of Mice

IntroductionHMG‐CoA reductase is a membrane protein of the endoplasmic reticulum (ER) that catalyzes the reduction of HMG‐CoA to mevalonate, a rate‐limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Sterol and nonsterol isoprenoids exert stringent feedback control on HMGCR through multiple mechanisms. This ensures constant synthesis of essential nonsterol isoprenoids, while avoiding toxic overaccumulation of cholesterol. One of these mechanisms involves sterol‐induced ubiquitination of HMGCR, which marks the enzyme for degradation from ER membranes that is augmented by nonsterol isoprenoids. In this study, we examine the contribution of this sterol‐accelerated ubiquitination/degradation to overall regulation of HMGCR in the livers of mice.MethodsForty mice, including 20 wild‐type (WT) and 20 knock‐in (Ki) mice that express ubiquitination‐resistant HMGCR, were fed diets containing only chow, or chow supplemented with 0.1%, 0.3%, or 1% cholesterol. After five days of feeding, livers were harvested for measurements of cholesterol and triglycerides, immunoblot analysis of six proteins, and qRTPCR of 26 genes related to cholesterol, nonsterol isoprenoid, and fatty acid synthesis.ResultsNormalization of mRNA levels to protein levels indicates that HMGCR Ki mouse livers contain a higher level of HMGCR protein despite mRNA downregulation. Protein and gene expression of SREBP‐2 and its target genes, which contribute to cholesterol synthesis, decreased as expected with increased dietary cholesterol. Conversely, protein and gene expression of SREBP‐1 and its target genes increased, likely due to SREBP‐1c predominance toward fatty acid synthesis, which prevents cholesterol accumulation.ConclusionThe increase in HMGCR protein relative to mRNA suggests that significant posttranscriptional regulation exists in the form of impaired degradation. Furthermore, these normalized values indicate that accumulation of protein is primarily due to impaired degradation at lower cholesterol levels (chow, 0.1%); however, at high cholesterol levels (0.3%, 1%), a greater degree of transcriptional control from sterol‐mediated inhibition of SREBP‐2 regulates HMGCR due to negative feedback. This study demonstrates the role of degradative control on inhibition of HMGCR and may assist in reducing HMGCR accumulation during statin therapy.Support or Funding InformationNIH grants HL020948, GM112409, Ruth L. Kirschstein National Research Service Award (NRSA) Short‐Term Institutional Research Training Grant (T‐35)