Sterol Regulatory Element-binding Transcription Factor Research Articles

Proteomic Analysis of Porcine Pre-ovulatory Follicle Differentiation Into Corpus Luteum.

The luteinization of the follicular cells, following a LH surge, causes extensive molecular and structural changes in preovulatory follicles (POF) that lead to ovulation and ultimate formation of the corpus luteum (CL). The objective of this study was to identify proteins expressed in porcine POF before the LH surge and a new CL formed, 2–3 days after ovulation, and evaluate proteome changes associated with formation of the CL from a follicle. We used 2D-gel electrophoresis-based proteomics and tandem mass spectrometry followed by a functional analysis using Ingenuity Pathway analysis (IPA) to evaluate functional pathways associated with the luteinization process. Protein lysates were prepared from isolated POFs and from the newly formed CL. A total of 422 protein spots were identified in both structures. A total of 15 and 48 proteins or their proteoforms were detected only in the POFs and CL, respectively. An IPA analysis of a POF proteome showed that most of the follicular proteins were involved in cellular infiltration, endoplasmic stress responses, and the protein ubiquitination pathway. Most of the early luteal proteins were associated with steroid metabolism, cell death and survival, free radical scavenging, and the protein ubiquitination pathway. A comparison of a follicular proteome with that of an early luteal proteome revealed that 167 identified proteins or their proteoforms were differentially regulated between POFs and the newly formed CL (p < 0.05 and a fold change of >1.8). Proteins that were significantly more abundant in follicles included cAMP-dependent protein kinase, histone binding protein RBBP4, reticulocalbin, vimentin, and calumenin; more abundant luteal proteins included albumin, farnesyl diphosphate synthase, serine protease inhibitors, elongation factor-1, glutaredoxin, and selenium-binding protein. Proteins that were significantly altered with luteal formation were found to be associated with cholesterol biosynthesis, cell death and survival, and acute phase response. Moreover, upstream regulators of differentially abundant proteins in CL were identified that included insulin growth factor-1, sterol regulatory element-binding transcription factor-1, and nuclear factor erythroid-derived 2. We have identified novel proteins that advance our understanding of (1) processes associated with differentiation of POFs into the CL, (2) possible mechanisms of luteal cell survival, and (3) pathways regulating steroidogenesis in the newly formed CL.

Disrupted Blood-Retina Lysophosphatidylcholine Transport Impairs Photoreceptor Health But Not Visual Signal Transduction.

Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood-retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using the Mfsd2a knock-out mouse as animal model. Our lipidome analyses of Mfsd2a-/- retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated in Mfsd2a-/- retinas. Mfsd2a-/- retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructure Mfsd2a-/- photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENT Phospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood-retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.

Allium hookeri Root Extract Inhibits Adipogenesis by Promoting Lipolysis in High Fat Diet-Induced Obese Mice.

Allium hookeri (AH) is widely consumed as a herbal medicine. It possesses biological activity against metabolic diseases. The objective of this study was to investigate effects of AH root water extract (AHR) on adipogenesis in 3T3-L1 cells and in high-fat diet (HFD)-induced obese mice. AHR inhibited lipid accumulation during adipocyte differentiation by downregulation of gene expression, such as hormone sensitive lipase (HSL), lipoprotein lipase (LPL) and an adipogenic gene, CCAAT/enhancer binding protein-α in 3T3-L1 preadipocytes. Oral administration of AHR significantly suppressed body weight gain, adipose tissue weight, serum leptin levels, and adipocyte cell size in HFD-induced obese mice. Moreover, AHR significantly decreased hepatic mRNA expression levels of cholesterol synthesis genes, such as 3-hydroxy-3-methylglutaryl CoA reductase, sterol regulatory element-binding transcription factor (SREBP)-2, and low-density lipoprotein receptor, as well as fatty acid synthesis genes, such as SREBP-1c and fatty acid synthase. Serum triglyceride levels were also lowered by AHR, likely as a result of the upregulating gene involved in fatty acid β-oxidation, carnitine palmitoyltransferase 1a, in the liver. AHR treatment activated gene expression of peroxisome proliferator-activated receptor-γ, which might have promoted HSL and LPL-medicated lipolysis, thereby reducing white adipose tissue weight. In conclusion, AHR treatment can improve metabolic alterations induced by HFD in mice by modifying expression levels of genes involved in adipogenesis, lipogenesis, and lipolysis in the white adipose tissue and liver.

Promoting differentiation and lipid metabolism are the primary effects for DINP exposure on 3T3-L1 preadipocytes

Diisononyl phthalate (DINP) is a high-molecular-weight phthalate, and has been recently introduced as di-(2-ethyl hexyl) phthalate (DEHP) substitute and commonly used in a large variety of plastic items. The fat tissue is an important target for DINP exposure, however, very little is understood about its toxicity and mechanism(s) in adipocyte cells. Therefore, the present work aimed to investigate the role of DINP in adipogenesis using 3T3-L1 preadipocytes. DINP exposure for 10 days extensively induced adipogenesis in 3T3-L1 preadipocytes to adipocytes as assessed by lipid accumulation and gene expression of adipogenic markers. The RT-qPCR results showed that DINP could upregulate the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα) and C/EBPβ, while the expression of sterol regulatory element binding transcription factor 1 (SREBF1) and C/EBPδ was not affected. The DINP-induced adipogenesis could be inhibited by using the selective PPARγ antagonist GW9662. The RNA-seq analysis was used to study the systemic toxicities of DINP on preadipocytes. A total of 1181 differently expressed genes (DEGs) (640 genes were up-regulated, 541 genes were down-regulated) were detected in 3T3-L1 preadipocytes under 50 μM DINP. The GO enrichment showed the GO term of “fat cell differentiation” was the most significantly affected metabolic functions, and the KEGG pathway enrichment showed the PPAR pathway was the top affected pathway. The interactive pathway (iPath) analysis showed that the changed metabolic pathways were focus on the lipid metabolism.

SREBF1c and SREBF2 gene polymorphisms are associated with acute coronary syndrome and blood lipid levels in Mexican population.

AimIt has recently been reported that the sterol regulatory element-binding transcription factors (SREBF-1c, and -2) contribute to the variation in the plasma lipids levels, which have an important role in the atherosclerotic plaque development. The aim of the present study was to evaluate whether the SREBF1c and SREBF2 gene single nucleotide polymorphisms (SNPs) are associated with plasma lipids levels and ACS susceptibility in a case-control association study.Material and methodsA case-control study was carried out in 625 patients with ACS (82% men and 18% women, with a mean age of 57.97 ± 10.5 years) and 700 healthy controls (66% men and 34% women, with a mean age of 54.37 ± 7.65 years). The sample size was calculated for a statistical power of 80%. We genotyped three SREBF1c (rs2297508, rs11656665 and rs11868035) and three SREBF2 (rs2267439, rs2267443, and rs2228314) gene polymorphisms by 5’ exonuclease TaqMan assays. The associations were evaluated by logistic regression under the co-dominant, dominant, recessive, over-dominant and additive inheritance models. The contribution of the genotypes on the plasma lipids levels was evaluated by Student’s t-test.ResultsUnder different models, the SREBF1c rs2297508 (OR = 1.50, pCRes = 0.03), SREBF1c rs11656665 (OR = 1.35, pCDom = 0.02 and OR = 1.26, pCAdd = 0.02) and SREBF2 rs2228314 (OR = 1.78, pCRes = 0.03, OR = 1.27, pCAdd = 0.04) SNPs were associated with higher risk of ACS. On the other hand, the SREBF1c rs11868035 SNP was associated with lower risk of ACS (OR = 0.49, pCCo-dom = 0.001, OR = 0.66, pCDom = 0.003, OR = 0.57, PRes = 0.003 and OR = 0.71, pCAdd = 0.001). There was a statistically significant association of both SREBF1c rs11656665 and rs11868035 polymorphisms with plasma triglyceride levels.ConclusionsIn summary, our data suggest the association of the SREBF1c and SREBF2 SNPs with risk of developing ACS and with triglyceride levels in a Mexican population.

Protective Effects of Lanosterol Synthase Up-Regulation in UV-B-Induced Oxidative Stress.

UV-B radiation may be an important risk factor in cataract etiology. After exposure to UV-B radiation, cells show imbalances in the repair of DNA damage, which induce changes in the levels of certain proteins, including alpha-crystallin, which is the most abundant protein in the lens and crucial for the maintenance of lens transparency. Lanosterol synthase (LSS), an essential rate-limiting enzyme in cholesterol biosynthesis, might play significant roles in oxidative stress and in the maintenance of lens transparency. However, the roles of LSS in UV-B-induced apoptosis are not well understood. Therefore, we irradiated female Sprague-Dawley rats with ultraviolet radiation to establish an animal model for exploring the variations in LSS expression during the early stages of UV-B exposure. In addition, we cultured human lens epithelial (HLE) cells that overexpress LSS and exposed them to UV-B radiation to explore the function of increased LSS expression in UV-B-induced apoptosis. The data demonstrated that UV-B exposure induced oxidative stress and apoptosis in rat lens epithelial cells and that irradiance exposure increased the level of lenticular damage. Additionally, UV-B exposure decreased the alpha-crystallin content and increased the expressions of Bax and cleaved caspase-3 compared with the control levels. After exposure to UV-B, the apoptosis-related index of HLE cells overexpressing LSS was lower than that of the control cells. Furthermore, ROS overproduction might activate the sirtuin 1 (Sirt1) pathway, which induced protein expressions of sterol regulatory element-binding transcription factor 2 (SREBF2), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), and LSS. However, the specific mechanism of the Sirt1 pathway needed to be further studied. In summary, UV-B exposure induced oxidative injury and resulted in crystallin denaturation and apoptosis in lens epithelial cells, and LSS might play a protective role during the early stages of this process and could be an important target in the cataract prevention.

Short-Term Oral UMP/UR Administration Regulates Lipid Metabolism in Early-Weaned Piglets.

Simple SummaryUridine monophosphate (UMP) and uridine (UR) are rich in sow’s milk. The results from this study showed that UMP and UR affect the lipid profile and lipid metabolism in weanling piglets. It is suggested that UMP and UR improve the energy status in early-weaned piglets.As a main ingredient of milk, the nucleotides content is about 12–58 mg/g, which plays a critical role in maintaining cellular function and lipid metabolism. This study was conducted to evaluate the effects of short-term uridine monophosphate (UMP) and uridine (UR) administration on lipid metabolism in early-weaned piglets. Twenty-one weaned piglets (7 d of age; 3.32 ± 0.20 kg average body weight) were randomly assigned into three groups: The control (CON), UMP, and UR group, and oral administered UMP or UR for 10 days, respectively. The results showed that supplementation with UMP significantly increased (p < 0.05) serum low density lipoprotein (LDL) and tended to increase (p = 0.062) serum total cholesterol (TC) content of piglets when compared with the other two groups. Oral administration with UMP and UR significantly decreased (p < 0.05) the serum total bile acid (TBA) and plasma free fatty acids (FFA) of piglets, and significantly reduced the fatty acid content of C12:0 (p < 0.01) and C14:0 (p < 0.05) in liver. Experiments about key enzymes that are involved in de novo synthesis of fatty acid showed that the gene expression of liver X receptors (LXRα), sterol regulatory element-binding transcription factor 1 (SREBP1c), fatty acid desaturase 2 (FADS2), and fatty acid elongase 5 (ELOVL5) were remarkably down-regulated (p < 0.05) with UMP and UR treatment, and key factors of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and carnitine palmitoyl transferase 1 (CPT-1α) involved in fatty acid catabolism were also decreased (p < 0.05). Additionally, the protein expression of phosphorylated-mTOR was not affected while phosphorylation of AKT was repressed (p < 0.05). In conclusion, short-term oral UMP or UR administration could regulate fatty acid composition and lipid metabolism, thus providing energy for early-weaned piglets.

Nutritional intervention through dietary vegetable proteins and lipids to gilthead sea bream (Sparus aurata) broodstock affects the offspring utilization of a low fishmeal/fish oil diet

Nutritional intervention with vegetable oils (VO) supplemented to fish broodstock diets enhances progeny ability to utilize low fish meal (FM) and fish oil (FO) diets. However, the intervention with vegetable meals (VM) in broodstock diets has been scarcely tested in marine fish. This study aims to determine the effect of FM replacement by VM alone or in combination with FO replacement by VO as a programming tool. Different broodstocks of gilthead sea bream were fed one of three diets: one contained 35% FM and 10% FO, another contained VM in replacement of FM (FM: 15%) or a third one had both VM and VO in replacement of FM and FO (15% FM and 2.7% FO). At the weight of 3 g, their offspring were challenged with a low FM (5%) and FO (3%) diet for 45 days. FM replacement by VM in broodstock diets did not affect fish reproductive performance but in offspring juveniles reduced growth and feed efficiency and in liver, altered fatty acids profiles and down-regulated fatty acyl desaturase 2 (fads2) expression. Combined supplementation of VM and VO in broodstock diets led to a poor reproductive performance, reducing female fecundity and the egg content in eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). At molecular level, the expression of fads2 and target of rapamycine (tor) genes in the eggs were upregulated. Similarly, in liver of juvenile progenies fads2 and elongase of very long chain fatty acids 6 (elovl6) expression were up-regulated, whereas fads2 expression was correlated to sterol regulatory element-binding protein transcription factor 1 (srebp1). However, methylation rates of CpG rich regions in fads2 promoter were not significantly different among juvenile groups. Thus, the replacement of FM by VM, alone or in combination with FO in diets for gilthead sea bream broodstock negatively affected growth performance of juvenile offspring, whereas the replacement of FO by VO enhanced the fatty acid desaturase and elongase activities in the offspring, especially when juveniles were fed a diet with a high replacement of FM and FO by VM and VO.

Oleacein Prevents High Fat Diet-Induced Adiposity and Ameliorates Some Biochemical Parameters of Insulin Sensitivity in Mice.

Oleacein is one of the most abundant polyphenolic compounds of olive oil, which has been shown to play a protective role against several metabolic abnormalities, including dyslipidemia, insulin resistance, and glucose intolerance. Herein, we investigated the effects of oleacein on certain markers of adipogenesis and insulin-resistance in vitro, in 3T3-L1 adipocytes, and in vivo in high-fat diet (HFD)-fed mice. During the differentiation process of 3T3-L1 preadipocytes into adipocytes, oleacein strongly inhibited lipid accumulation, and decreased protein levels of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid synthase (FAS), while increasing Adiponectin levels. In vivo, treatment with oleacein of C57BL/6JOlaHsd mice fed with HFD for 5 and 13 weeks prevented the increase in adipocyte size and reduced the inflammatory infiltration of macrophages and lymphocytes in adipose tissue. These effects were accompanied by changes in the expression of adipose tissue-specific regulatory elements such as PPARγ, FAS, sterol regulatory element-binding transcription factor-1 (SREBP-1), and Adiponectin, while the expression of insulin-sensitive muscle/fat glucose transporter Glut-4 was restored in HFD-fed mice treated with oleacein. Collectively, our findings indicate that protection against HFD-induced adiposity by oleacein in mice is mediated by the modulation of regulators of adipogenesis. Protection against HFD-induced obesity is effective in improving peripheral insulin sensitivity.

MiR-142-3p Regulates Milk Synthesis and Structure of Murine Mammary Glands via PRLR-Mediated Multiple Signaling Pathways.

Murine mammary gland is an ideal model for studying the development and milk synthesis in dairy animals. MicroRNAs play an important role in milk synthesis and mammary gland development; however, the molecular mechanism of miR-142-3p continues to be poorly understood. Here, we knocked down miR-142-3p expression in vitro and vivo, increased the prolactin receptor expression and activated many downstream cellular proteins, such as mammalian target of rapamycin, sterol regulatory element-binding transcription factor 1, cyclin D1, and signal transducer and activator of transcription 5. Additionally, miR-142-3p knockdown in mouse mammary gland epithelial cells increased proliferation but not viability, induced cell cycle progression, decreased apoptosis, and increased the expression of triglycerides and β-casein. Moreover, miR-142-3p knockdown in murine mammary gland tissue in vivo affected the structure and function of the mammary gland, which showed an increased number of lobules and ducts and was more capable of producing milk. However, overexpression of miR-142-3p had the opposite effects. In summary, these data reveal that miR-142-3p regulates milk synthesis and the structure of murine mammary glands via PRLR-mediated multiple signaling pathways.

OSBPL2 deficiency upregulate SQLE expression increasing intracellular cholesterol and cholesteryl ester by AMPK/SP1 and SREBF2 signalling pathway

Previous studies have shown that oxysterol binding protein like 2 (OSBPL2) knockdown is closely related to cholesterol metabolism. However, whether there is a direct relation between OSBPL2 and cholesterol synthesis is unknown. This study explored the mechanism of OSBPL2 deficiency in the upregulation of squalene epoxidase (SQLE) and the subsequent accumulation of intracellular cholesterol and cholesteryl ester. Here, we constructed an OSBPL2-deleted HeLa cell line using CRISPR/Cas9 technology, screened differentially expressed genes and examined the transcriptional regulation of SQLE using a dual-luciferase reporter gene. RNA-seq analysis showed that SQLE was upregulated significantly and the dual luciferase reporter gene assay revealed that two new functional transcription factor binding sites of Sp1 transcription factor (SP1) and sterol regulatory element-binding transcription factor 2 (SREBF2) in the SQLE promoter participated in the SQLE transcription and expression. In addition, we also observed that OSBPL2 deletion inhibited the AMPK signalling pathway and that the inhibition of AMPK signalling promoted SP1 and SREBF2 entry into the nuclear to upregulate SQLE expression. Therefore, these data support that OSBPL2 deficiency upregulates SQLE expression and increases the accumulation of cholesterol and cholesteryl ester by suppressing AMPK signalling, which provides new evidence of the connection between OSBPL2 and cholesterol synthesis.

Isoimperatorin enhances 3T3-L1 preadipocyte differentiation by regulating PPARγ and C/EBPα through the Akt signaling pathway.

Lipodystrophic patients have an adipose tissue triglyceride storage defect that causes ectopic lipid accumulation, leading to severe insulin resistance. The present study investigated the potential role of isoimperatorin on 3T3-L1 adipocyte differentiation. mRNA and protein levels of differentiation- and lipid accumulation-associated genes, as well as the adipogenesis-related signaling pathway were analyzed in control and isoimperatorin-treated differentiated 3T3-L1 adipocytes using reverse transcription-quantitative PCR and western blot analysis. Results determined that isoimperatorin promoted 3T3-L1 fibroblast adipogenesis in a dose-dependent manner compared with standard differentiation inducers. Isoimperatorin significantly increased mRNA and protein expression of the crucial adipogenic transcription factors peroxisome proliferator activated receptor-γ (PPARγ) and CCAAT enhancer binding protein-α (C/EBPα). mRNA expression of the downstream adipogenesis-related genes sterol regulatory element-binding transcription factor 1c, adipocyte protein 2, fatty acid synthase, adiponectin and diacylglycerol O-acyltransferase 2 were also significantly increased following isoimperatorin treatment. The underlying mechanism likely involved activation of the Akt signaling pathway. Taken together, the present findings indicated that isoimperatorin may alter PPARγ and C/EBPα expression via the Akt signaling pathway, resulting in promotion of adipogenesis. The results highlighted the potential use of isoimperatorin as a therapeutic agent for preventing diabetes.

Effect of divergence in residual feed intake on expression of lipid metabolism-related genes in the liver of meat-type ducks1

Lipid metabolism is considered one of the important factors affecting residual feed intake (RFI). However, the relationship between RFI and expression of lipid metabolism-related genes is unknown in meat-type ducks. To address this issue, a total of 1,000 male meat-type ducks with similar body weight were randomly selected to measure body weight gain and feed intake from 21 to 42 d of age to estimate RFI. The 8 greatest- (high RFI [HRFI]) and lowest- (low RFI [LRFI]) ranking birds were then selected for the present study. Relative expressions of key genes, namely sirtuin 1 (Sirt1), forkhead box O1 (Foxo1), peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1c (SREBP-1c), fas cell surface death receptor (FAS), acetyl-CoA carboxylase alpha (ACC), carnitine palmitoyltransferase 1A (CPT1A), and acyl-CoA oxidase 1 (ACOX1), were then determined in the HRFI and LRFI ducks by quantitative PCR. The results showed that RFI, feed conversion ratio (FCR), and average daily feed intake (ADFI) were significantly lower (P < 0.05) in LRFI ducks than in HRFI ducks. In addition, expression of Sirt1, Foxo1, CPT1A, and ACOX1 were significantly higher in LRFI ducks than in HRFI ducks (P < 0.05), whereas PPARγ and FAS expression levels were significantly lower in LRFI ducks than in HRFI ducks (P < 0.01). Correlation analysis showed that Sirt1, CPT1A, and ACOX1 expressions were significantly negatively correlated with FCR (r = -0.81 to -0.93; P < 0.01), whereas PPARγ and FAS expressions were significantly positively correlated with FCR (r = 0.74 to 0.87; P < 0.01). PPARγ expression was significantly positively correlated with RFI (r = 0.83; P < 0.01), whereas CPT1A and ACOX1 expressions were significantly negatively correlated with RFI (r = -0.84 to -0.89; P < 0.01). Sirt1 mRNA expression was positively correlated with Foxo1, CPT1A, and ACOX1 mRNA expression (r = 0.78 to 0.92; P < 0.01). Association of Foxo1 with CPT1A and ACOX1 was positive (r = 0.88 to 0.96; P < 0.01). These results suggest that genes related to fatty acid oxidation are upregulated in the liver of ducks with high feed efficiency, while genes associated with lipid synthesis are downregulated. Furthermore, the inclusion of lipid metabolism-related genes in future breeding programs might be beneficial for selecting ducks with greater feed efficiency phenotype.

MTORC2 Deficiency Alters the Metabolic Profile of Conventional Dendritic Cells.

In myeloid dendritic cells (DC), deletion of the mechanistic target of rapamycin complex 2 (TORC2) results in an augmented pro-inflammatory phenotype and T cell stimulatory activity; however, the underlying mechanism has not been resolved. Here, we demonstrate that mouse bone marrow-derived TORC2-deficient myeloid DC (TORC2−/− DC) utilize an altered metabolic program, characterized by enhanced baseline glycolytic function compared to wild-type WT control (Ctrl) DC, increased dependence on glycolytic ATP production, elevated lipid content and higher viability following stimulation with LPS. In addition, TORC2−/− DC display an increased spare respiratory capacity (SRC) compared to WT Ctrl DC; this metabolic phenotype corresponds with increased mitochondrial mass and mean mitochondrial DNA copy number, and failure of TORC2−/− DC mitochondria to depolarize following LPS stimulation. Our data suggest that the enhanced metabolic activity of TORC2−/− DC may be due to compensatory TORC1 pathway activity, namely increased expression of multiple genes upstream of Akt/TORC1 activity, including the integrin alpha IIb, protein tyrosine kinase 2/focal adhesion kinase, IL-7R and Janus kinase 1(JAK1), and the activation of downstream targets of TORC1, including p70S6K, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and CD36 (fatty acid translocase). These enhanced TORC1 pathway activities may culminate in increased expression of the nuclear receptor peroxisome proliferator-activated receptor γ (Pparγ) that regulates fatty acid storage, and the transcription factor sterol regulatory element-binding transcription factor 1 (Srebf1). Taken together, our data suggest that TORC2 may function to restrain TORC1-driven metabolic activity and mitochondrial regulation in myeloid DC.

Abstract 1829: HSF1 suppresses AMPK to promote lipogenesis, cholesterol biosynthesis and protein lipidation

Abstract AMP-activated protein kinase (AMPK), a key cellular metabolic sensor, suppresses anabolic pathways, including lipogenesis and cholesterol biosynthesis, thereby maintaining energy homeostasis. Heat shock factor 1 (HSF1) is the master regulator of the evolutionarily conserved proteotoxic stress response (PSR), thereby preserving proteostasis in the face of environmental insults. In contrast to its beneficial role in enhancing survival under proteotoxic stress, it has been demonstrated that HSF1 acts as a potent pro-oncogenic factor. Our previous study has shown that metabolic stressors, via AMPK activation, inactivate HSF1 and disrupt proteostasis to suppress tumor growth. Interestingly, our new study now reveals that HSF1, in reciprocal, also suppresses AMPK activation. HSF1 does so independently of its canonical transcriptional action. Furthermore, Hsf1 deficiency promotes the interactions between AMPK and LKB1, a key upstream kinase activating AMPK. Congruent with the suppression of lipogenesis by AMPK, Hsf1 deficiency depletes cellular lipid content and reduces body fat mass in mice. Importantly, these defects can be largely rescued by either pharmacological or genetic inhibition of AMPK. At the molecular level, Hsf1 deficiency leads to inactivation of acetyl-CoA Carboxylase (ACC) and sterol regulatory element-binding transcription factor 1c (SREBP1c), both of which are key players in de novo lipogenesis. In addition to impaired lipogenesis, Hsf1-deficient cells and mice display reduced cholesterol levels. Of particular interest, this defect leads to impaired cholesteroylation of sonic hedgehog (SHH), the best known signaling molecule undergoing this covalent modification. In consequence, SHH secreted by HSF1-deficient cells are less potent in activating its downstream signaling cascade, indicated by reduced DNA binding of GLI1, a transcription factor responding to activation of SHH signaling. Conversely, HSF1 overexpression increases the lipid content, cholesterol levels and the amount of cholesteroylated SHH proteins in xenografted melanomas. Taken together, our results reveal that HSF1, through AMPK suppression, promotes lipogenesis, cholesterol biosynthesis and protein lipidation, thereby supporting malignant growth. Citation Format: Kuo-Hui Su, Chengkai Dai. HSF1 suppresses AMPK to promote lipogenesis, cholesterol biosynthesis and protein lipidation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1829.

Apical sodium-dependent bile acid transporter inhibition with volixibat improves metabolic aspects and components of non-alcoholic steatohepatitis in Ldlr-/-.Leiden mice.

Interruption of bile acid recirculation through inhibition of the apical sodium-dependent bile acid transporter (ASBT) is a promising strategy to alleviate hepatic cholesterol accumulation in non-alcoholic steatohepatitis (NASH), and improve the metabolic aspects of the disease. Potential disease-attenuating effects of the ASBT inhibitor volixibat (5, 15, and 30 mg/kg) were investigated in high-fat diet (HFD)-fed Ldlr-/-.Leiden mice over 24 weeks. Plasma and fecal bile acid levels, plasma insulin, lipids, and liver enzymes were monitored. Final analyses included liver histology, intrahepatic lipids, mesenteric white adipose tissue mass, and liver gene profiling. Consistent with its mechanism of action, volixibat significantly increased the total amount of bile acid in feces. At the highest dose, volixibat significantly attenuated the HFD-induced increase in hepatocyte hypertrophy, hepatic triglyceride and cholesteryl ester levels, and mesenteric white adipose tissue deposition. Non-alcoholic fatty liver disease activity score (NAS) was significantly lower in volixibat-treated mice than in the HFD controls. Gene profiling showed that volixibat reversed the inhibitory effect of the HFD on metabolic master regulators, including peroxisome proliferator-activated receptor-γ coactivator-1β, insulin receptor, and sterol regulatory element-binding transcription factor 2. Volixibat may have beneficial effects on physiological and metabolic aspects of NASH pathophysiology.

Pharmacologic Inhibition of Epidermal Growth Factor Receptor Suppresses Nonalcoholic Fatty Liver Disease in a Murine Fast-Food Diet Model.

Epidermal growth factor receptor (EGFR) is a critical regulator of hepatocyte proliferation and liver regeneration. Our recent work indicated that EGFR can also regulate lipid metabolism during liver regeneration after partial hepatectomy. Based on these findings, we investigated the role of EGFR in a mouse model of nonalcoholic fatty liver disease (NAFLD) using a pharmacological inhibition strategy. C57BL6/J mice were fed a chow diet or a fast-food diet (FFD) with or without EGFR inhibitor (canertinib) for 2 months. EGFR inhibition completely prevented development of steatosis and liver injury in this model. In order to study if EGFR inhibition can reverse NAFLD progression, mice were fed the FFD for 5 months, with or without canertinib treatment for the last 5 weeks of the study. EGFR inhibition remarkably decreased steatosis, liver injury, and fibrosis and improved glucose tolerance. Microarray analysis revealed that ~40% of genes altered by the FFD were differentially expressed after EGFR inhibition and, thus, are potentially regulated by EGFR. Several genes and enzymes related to lipid metabolism (particularly fatty acid synthesis and lipolysis), which were disrupted by the FFD, were found to be modulated by EGFR. Several crucial transcription factors that play a central role in regulating these lipid metabolism genes during NAFLD, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBF1), carbohydrate-responsive element-binding protein, and hepatocyte nuclear factor 4 alpha, were also found to be modulated by EGFR. In fact, chromatin immunoprecipitation analysis revealed that PPARγ binding to several crucial lipid metabolism genes (fatty acid synthase, stearoyl-coenzyme A desaturase 1, and perilipin 2) was drastically reduced by EGFR inhibition. Further upstream, EGFR inhibition suppressed AKT signaling, which is known to control these transcription factors, including PPARγ and SREBF1, in NAFLD models. Lastly, the effect of EGFR in FFD-induced fatty-liver phenotype was not shared by receptor tyrosine kinase MET, investigated using MET knockout mice. Conclusion: Our study revealed a role of EGFR in NAFLD and the potential of EGFR inhibition as a treatment strategy for NAFLD.

Comparative omics analyses of hepatotoxicity induced by oral azole drugs in mice liver and primary hepatocytes

Ketoconazole (KTZ) and itraconazole (ITZ) are antifungal agents that have a broad spectrum of activity against fungal pathogens. However, the therapeutic indications of many antifungal drugs, including those of the azole group, are restricted due to possible hepatotoxicity. We performed toxicogenomic analyses using in vivo and in vitro models to investigate the molecular mechanisms underlying the hepatotoxicity of two azole antifungal drugs. C57BL/6 male mice were treated daily with KTZ or ITZ, sacrificed at days 1 or 7, and the serum biochemistry and histopathology results showed that the KTZ-treated mice exhibited hepatotoxicity. Primary hepatocytes from C57BL/6 mice also exposed to KTZ or ITZ, and the cytotoxic effects of KTZ and ITZ were evaluated; KTZ exerted a greater cytotoxic effect than ITZ. The gene expression profiles in the livers of the 7-day-treated group and primary hepatocytes of the 24-h-treated group for both KTZ and ITZ were comparatively analyzed. Differentially expressed genes were selected based on the fold-changes and statistical significance, and the biological functions were analyzed using ingenuity pathways analysis. The results revealed that genes related to cholesterol synthesis were overexpressed in the liver in the KTZ-treated group, whereas expression of those related to acute phase injury was significantly altered in the ITZ-treated group. Causal gene analyses suggested that sterol regulatory element-binding transcription factors are key regulators that activate the transcription of target genes associated with the hepatotoxicity induced by oral KTZ. These findings enhance our understanding of the molecular mechanisms underlying the hepatotoxicity of azole drugs.

Elevated maternal linoleic acid reduces circulating leptin concentrations, cholesterol levels and male fetal survival in a rat model.

Linoleic acid consumption is increasing in Western populations. We investigated whether elevated linoleic acid in pregnancy was deleterious to mothers or offspring. Maternal and fetal body and organ weights were not affected by elevated linoleic acid consumption. Maternal lipids and leptin were altered following elevated linoleic acid consumption. Male offspring numbers were reduced following elevated linoleic acid consumption. Dietary intakes of linoleic acid (LA) have increased dramatically in Western populations, including in women of reproductive age. Pro-inflammatory effects of LA may have detrimental effects on maternal and offspring outcomes. We aimed to investigate whether consumption of a maternal diet with elevated LA altered maternal inflammatory or metabolic markers during pregnancy, fetal growth and/or the sex ratio of the offspring. Female Wistar Kyoto rats consumed a diet high in LA (HLA) (6.21% of energy) or a diet low in LA (LLA) (1.44% of energy) for 10weeks prior to mating and during pregnancy. Pregnant rats were killed at embryonic day 20 (E20). There were no differences in maternal or fetal body weights or organ weights in the HLA group compared to the LLA group. There was no difference in maternal circulating cytokine concentrations between dietary groups. In the maternal liver, IL-1α concentrations were significantly lower, and TNF-α and IL-7 significantly higher in the HLA group. Total plasma cholesterol, LDL-cholesterol, HDL cholesterol and the total:HDL cholesterol ratio were lower in dams fed the HLA diet. mRNA expression of sterol regulatory element binding transcription factor 1 (SREBF-1) and leptin in maternal adipose tissue was lower in the HLA group, as were circulating leptin concentrations. The proportion of male fetuses was lower and circulating prostaglandin E metabolite concentrations were increased in the HLA group. In conclusion, consumption of a maternal diet high in linoleic acid alters cholesterol metabolism and prostaglandin E metabolite concentrations, which may contribute to the reduced proportion of male offspring.

Thermoneutrality Inhibits Thermogenic Markers and Exacerbates Nonalcoholic Fatty Liver in Mice (P21-068-19)

ObjectivesNonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease, affecting more than a third of the US population and 25% of the global population. In this study, we investigated whether manipulation of brown adipose tissue (BAT) activity via housing C57BL/6 J mice under two different housing temperatures (22°C vs 27°C) influences the development of NAFLD. MethodsMale C57BL/6 J mice were randomly allocated to 4 groups (8 mice/group). Briefly, mice housed at the standard temperature (ST) (22°C) or thermoneutral temperature (TT) (27°C) were fed either a control chow diet (CHD) (Picolab rodent diet 20, LabDiet) with a regular drinking water or an “fast food” diet (FFD) (Research Diets, D12079B) plus fructose 23.1 g/L and glucose 18.9 g/L added to the drinking water for 10 weeks. ResultsMice under TT had significant less food intake compared to their counter partners under ST, regardless of diets. Mice exposed to TT and fed FFD had the highest body weight and plasma leptin level among all groups. For mice fed CHD, mice at TT did not present hepatic steatosis although they slightly gained more body weight compared to mice at ST. For mice fed FFD, thermoneutral housing mice had greater liver weight, liver triglyceride, and exacerbated hepatic steatosis compared to the standard housing mice. Moreover, for mice fed FFD, TT compared to ST had significantly elevated expression of fatty acid synthase, sterol regulatory element-binding transcription factor 1, fatty acid translocase, and monocyte chemoattractant protein-1 in liver. By contrast, TT did not change expression of above genes in mice fed CHD. Furthermore, thermoneutrally housed mice displayed a decrease in gene expression of thermogenic markers, such as uncoupling protein 1, elongation of very long chain fatty acids 3, and cell death-inducing DNA fragmentation factor alpha-like effector A in BAT and inguinal white adipose tissue (WAT). ConclusionsThermoneutrality inhibits expression of thermogenic markers in both BAT and WAT, which correlated to exacerbated NAFLD in mice. Our data indicate that activating BAT and/or promoting WAT browning may represent a potential strategy for the management of NAFLD. Funding SourcesThe College of Human Sciences of Texas Tech University; the National Center for Complementary & Integrative Health.