DGAT2 Expression Research Articles

Elongase of very long chain fatty acids 6 (ELOVL6) promotes lipid synthesis in buffalo mammary epithelial cells.

Recent studies have shown elongase of very-long-chain fatty acids 6 (ELOVL6) is a vital protein for endogenous synthesis of saturated and monounsaturated long-chain fatty acids in some mammals. Nevertheless, its role in lipid synthesis in buffalo mammary gland is still unclear. In this work, the full-length coding sequence (CDS) of ELOVL6 was cloned and identified from buffalo mammary gland. As a result, the CDS of this gene is 795bp, which encodes a polypeptide of 264 amino acid residues. The buffalo ELOVL6 contains an ELO domain which belongs to the ELO superfamily. Among the 10 tissues of buffalo in peak lactation detected by RT-qPCR, the expression level of ELOVL6 was the highest in the brain, followed by the spleen, and then decreased in the mammary gland, muscle, kidney, heart, liver, rumen, intestine and lung. However, only the expression in the brain and spleen was statistically different from that in other tissues (p<0.05). Compared with that of the dry-off period, the mRNA abundance of ELOVL6 in the mammary gland was significantly increased in peak lactation. The experiments based on lentivirus transfection in buffalo mammary epithelial cells (BuMECs) displayed that the overexpression of ELOVL6 markedly promoted the expression of INSIG1, INSIG2, SREBP, PPARG, FASN, GPAM, DGAT2 and APGAT6 genes, and the knockdown of ELOVL6 significantly decreased the mRNA abundance of INSIG2, SREBP, FASN, SCD, GPAM, APGAT6 and TIP47 genes. In addition, the increase or decrease of ELOVL6 expression level also caused the corresponding change of total triglyceride content in the BuMECs. The results here suggest that the ELOVL6 can catalyse the synthesis of long-chain fatty acids in the BuMECs, and it can indirectly affect the expression of genes related to milk fat synthesis through its catalytic products to promote the lipid biosynthesis of BuMECs.

Up-regulation of DGAT1 in cancer tissues and tumor-infiltrating macrophages influenced survival of patients with gastric cancer

BackgroundDiacylglycerol-acyltransferase 1 (DGAT1) plays an important role in the energy storage and is involved in cancer progression. A growing number of evidences showed that elevated expression of DGAT1 in cancer tissue indicated a poor outcome in cancer patients. However, the relationship between DGAT1 and gastric cancer is still unclear. Thus, Transcriptomic analysis and in vitro experiments were performed to investigate the role of DGAT1 in gastric cancer, as well as the potential therapy target in gastric cancer treatment.MethodsWe screened the public cancer datasets to identify the expression and function of DGAT1 in gastric cancer and tumor infiltrating lymphocytes. Then we testified the DGAT1 expression and function after sodium oleate treatment in AGS and MKN45 cell line. Finally, we analyzed ration of apoptosis, necrosis in gastric cancer cells by using flow cytometry after administration of DGAT1 inhibitor.ResultsOur results showed a highly expression of DGAT1 in gastric cancer tissues (n = 5, p = 0.0004), and tumor-infiltrating macrophages with elevated DGAT1 expression is associated with poor overall survival in gastric cancer patients. In addition, gastric cell lines AGS (n = 3, p < 0.05) and MKN45 (n = 3, p < 0.01) expressed higher level of DGAT1 than human gastric mucosal epithelial cell line GES-1. Administration of DGAT1 inhibitor effectively suppressed functional factors expression and induced cell death in MKN45.ConclusionThe findings of this research provide an in-depth insight into the potential role and influences involved in DGAT1 in the gastric cancer patients. And higher expression of DGAT1 leads to lower overall survival (OS) rate in patients with poorly differentiated gastric cancer. Our findings suggest a potential role for DGAT1 in the gastric cancer progression and inhibiting DGAT1 might be a promising strategy in gastric cancer treatment.

Alteration of lipid metabolism, autophagy, apoptosis and immune response in the liver of common carp (Cyprinus carpio) after long-term exposure to bisphenol A

Bisphenol A (BPA), as a phenolic compound, is harmful to human health, and its residue in the aquatic environment also threatens the health of aquatic animals. In this research, the toxicity effects of BPA on liver tissues were evaluated in common carp (Cyprinus carpio) after long-term exposure. Fish were exposed to five concentrations of BPA (0, 0.01, 0.1, 0.5 and 2 mg/L) for 30 days. The blood and liver tissues were gathered to analyze biochemical indices and genes transcription levels. The data related to lipid metabolism showed that BPA exposure increased serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) levels, upregulated the expressions of fatp1, pparγ, fas, atgl, hsl, pparα, cpt1b, acox-1, and downregulated the expression of dgat1 in liver. Antioxidative parameters displayed a reduced antioxidant ability and increased lipid peroxidation after BPA exposure. Meanwhile, the upregulations of nrf2, ho-1, cyp1a and cyp1b genes revealed an adaptive response mechanism against oxidative stress-induced adverse effects. After 30 days of exposure, BPA induced apoptosis and endoplasmic reticulum stress (ERS) via upregulating the expression levels of apoptosis and ERS-related genes and increasing Ca2+ concentration in liver. Moreover, the downregulation of mtor and the upregulation of atg3, atg7, tfeb, uvrag and mcoln1 indicated that BPA could influence the normal process of autophagy. Furthermore, BPA exposure activated toll like receptors (TLRs) pathway to mediate the inflammatory response. Our results demonstrated that BPA exposure disturbed lipid metabolism, and induced oxidative stress, ERS, apoptosis, autophagy and inflammatory response in the liver of common carp. These findings contributed to the understanding of hepatotoxicity mechanism induced by BPA in fish.

Peroxisome proliferator-activated receptor gamma regulates genes involved in milk fat synthesis in mammary epithelial cells of water buffalo.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical transcription factor regulating lipid and glucose metabolism. However, the regulatory effect of PPARγ on milk fat synthesis in buffalo mammary gland is not clear. In order to explore the role of buffalo PPARG gene in milk fat synthesis, lentivirus-mediated interference was used to knock it down and then the recombinant fusion expression vector was transfected into buffalo mammary epithelial cell (BMEC) to overexpress it. PPARG gene knockdown significantly decreased the expression of CD36, FABP3, FABP4, ACSS2, ELOVL6, DGAT2, BTN1A1, AGPAT6, LPIN1, ABCG2, PPARGC1A, INSIG1, FASN, and SREBF2 genes and significantly upregulated the expression of INSIG2 gene but had no significant effect on the expression of ACSL1, GPAM, and SREBF1 genes. PPARG overexpression significantly increased the relative mRNA abundance of CD36, FABP3, FABP4, ACSS2, ELOVL6, DGAT2, BTN1A1, AGPAT6, LPIN1, PPARGC1A, INSIG1, and SREBF2 genes and significantly downregulated the expression of INSIG2 gene but had no significant effect on the expression of ACSL1, GPAM, ABCG2, FASN, and SREBF1 genes. In addition, knockdown/overexpression of PPARG gene significantly decreased/increased triacylglycerol (TAG) content in BMECs. This study revealed that buffalo PPARG gene is a key gene regulating buffalo milk fat synthesis.

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators.

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

Short communication: Locus-specific interrelations between gene expression and DNA methylation patterns in bovine mammary gland infected by coagulase-positive and coagulase-negative staphylococci

Pathogens are able to alter the cell cycle program and immune response of the host by changing the transcription and epigenetics of genes responsible for cell cycle control and inflammation. In this regard, we evaluated interrelations between DNA methylation and expression of autophagy, apoptosis, and lipid metabolism-related genes in a sample set of mammary gland secretory tissue sections derived from bovine mammary glands infected with coagulase-negative and coagulase-positive staphylococci. We assessed relative transcript abundance and DNA bisulfite sequencing in loci of the ATG5, IGF1R, TERT, and DGAT1 genes. Lack of DNA methylation in ATG5 and DGAT1 loci might be associated with maintenance of ATG5 and DGAT1 expression regardless of the health status of bovine mammary gland. Complete methylation of intragenic CpG regions in the IGF1R locus was apparently not related to the presence of its transcript in the investigated udder parenchyma samples. Detected hypermethylation of the TERT upstream element was associated with a small amount of TERT mRNA in bovine mammary gland, regardless of the presence, or absence, of the pathogen. A significant decrease in TERT gene expression in tissue sections of mammary gland free of bacteria and in those infected with coagulase-positive staphylococci was observed in parenchyma samples infected with coagulase-negative staphylococci. Two possible explanations are the direct involvement of the TERT gene in the etiology of bovine mastitis or the increase of TERT mRNA due to activation of the MAPK signaling pathway in response to release of exotoxins by coagulase-negative bacteria in the bovine mammary gland.

CTRP12 inhibits triglyceride synthesis and export in hepatocytes by suppressing HNF-4α and DGAT2 expression.

C1q/TNF-related protein 12 (CTRP12) is an antidiabetic adipokine whose circulating levels are reduced in obesity and diabetes. Although partial and complete loss-of-function mouse models suggest a role for CTRP12 in modulating lipid metabolism and adiposity, its effect on cellular lipid metabolism remains poorly defined. Here, we demonstrate a direct action of CTRP12 in regulating lipid synthesis and secretion. In hepatoma cells and primary mouse hepatocytes, CTRP12 treatment inhibits triglyceride synthesis by suppressing glycerophosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT) expression. CTRP12 treatment also downregulates the expression of hepatocyte nuclear factor-4α (HNF-4α) and its target gene microsomal triglyceride transfer protein (MTTP), leading to reduced very-low-density lipoprotein (VLDL)-triglyceride export from hepatocytes. Consistent with the invitro findings, overexpressing CTRP12 lowers fasting and postprandial serum triglyceride levels in mice. These results underscore the important function of CTRP12 in lipid metabolism in hepatocytes.

Effect of INSIG1 on the milk fat synthesis of buffalo mammary epithelial cells.

We hypothesized that insulin-induced gene 1 (INSIG1) affects milk fat synthesis in buffalo. For this reason, the protein abundance of INSIG1 in the mammary tissue of buffalo during the peak period of lactation and dry-off period was evaluated. The results showed that the expression of INSIG1 at the peak of lactation was lower than that in the dry-off period. To explore the role of INSIG1 in milk fat synthesis, the buffalo mammary epithelial cells (BMECs) were isolated and purified from buffalo mammary tissue, and INSIG1 gene were overexpressed and knocked down by constructing the recombinant lentivirus vector of INSIG1 gene and transfecting into BMECs. Results revealed that INSIG1 overexpression decreased the expression of INSIG2, SREBP, PPARG, SCD, GPAM, DGAT2 and AGPAT6, which led to reduction of triglycerides (TAG) content in the cell. In contrast, knockdown of INSIG1 had a positive effect on mRNA expression of the above genes. Overall, the data provide strong support for a key role of INSIG1 in the regulation of milk fat synthesis in BMECs.

Bromodomain-containing protein 4 regulates a cascade of lipid-accumulation-related genes at the transcriptional level in the 3T3-L1 white adipocyte-like cell line

Our previous study demonstrated that the transfection of a short hairpin (sh)RNA targeting bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) family of proteins, into 3T3-L1 cells, a white adipocyte-like cell line, reduced the expression of insulin sensitivity genes, such as Adipoq, Fabp4, Lpl, Slc2a4 and Dgat1, and that BRD4 directly bound to the Adipoq, Slc2a4 and Lpl genes. In the present study, we aimed to identify other target genes of BRD4 by microarray analysis of Brd4 shRNA- and control shRNA-transfected cells. We found that the expression of many genes related to fat metabolism, and particularly those involved in fat accumulation in the glycolytic pathway, tricarboxylic acid cycle, and triacylglycerol synthesis, such as Dgat2, Gpd1, Acsl1, Pnpla2, Pgkfb3, Pcx, Fasn, Acacb and Cidec, was reduced by Brd4 shRNA transfection 2 and 8 days after the end of adipocyte differentiation. The binding of BRD4 at the 2-day and histone acetylation at the 8-day time point, in the vicinity of the Dgat2, Gpd1, Acsl1 and Cidec genes, was also reduced by Brd4 shRNA transduction. Treatment with low doses (10–100 nM) of the BET family inhibitor (+)-JQ-1 for 2, 4 or 8 days also reduced the expression of Dgat2, Gpd1, Fasn, Acab, Acsl1, Pnpla2 and Cidec in 3T3-L1 white adipocyte-like cells. These results indicate that BRD4 regulates the expression of numerous genes involved in lipid accumulation at the transcriptional level in a white adipocyte-like cell line.

Cell-Type–Specific, Ketohexokinase-Dependent Induction by Fructose of Lipogenic Gene Expression in Mouse Small Intestine

High intakes of fructose are associated with metabolic diseases, including hypertriglyceridemia and intestinal tumor growth. Although small intestinal epithelia consist of many different cell types, express lipogenic genes, and convert dietary fructose to fatty acids, there is no information on the identity of the cell type(s) mediating this conversion and on the effects of fructose on lipogenic gene expression. We hypothesized that fructose regulates the intestinal expression of genes involved in lipid and apolipoprotein synthesis, that regulation depends on the fructose transporter solute carrier family 2 member a5 [Slc2a5 (glucose transporter 5)] and on ketohexokinase (Khk), and that regulation occurs only in enterocytes. We compared lipogenic gene expression among different organs from wild-type adult male C57BL mice consuming a standard vivarium nonpurified diet. We then gavaged twice daily for 2.5 d fructose or glucose solutions (15%, 0.3 mL per mouse) into wild-type, Slc2a5-knockout (KO), and Khk-KO mice with free access to the nonpurified diet and determined expression of representative lipogenic genes. Finally, from mice fed the nonpurified diet, we made organoids highly enriched in enterocyte, goblet, Paneth, or stem cells and then incubated them overnight in 10 mM fructose or glucose. Most lipogenic genes were significantly expressed in the intestine relative to the kidney, liver, lung, and skeletal muscle. In vivo expression of Srebf1, Acaca, Fasn, Scd1, Dgat1, Gk, Apoa4, and Apob mRNA and of Scd1 protein increased (P<0.05) by 3- to 20-fold in wild-type, but not in Slc2a5-KO and Khk-KO, mice gavaged with fructose. In vitro, Slc2a5- and Khk-dependent, fructose-induced increases, which ranged from 1.5- to 4-fold (P<0.05), in mRNA concentrations of all these genes were observed only in organoids enriched in enterocytes. Fructose specifically stimulates expression of mouse small intestinal genes for lipid and apolipoprotein synthesis. Secretory and stem cells seem incapable of transport- and metabolism-dependent lipogenesis, occurring only in absorptive enterocytes.

The Role of Bone Morphogenetic Protein Signaling in Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) affects over 30% of adults in the United States. Bone morphogenetic protein (BMP) signaling is known to contribute to hepatic fibrosis, but the role of BMP signaling in the development of NAFLD is unclear. In this study, treatment with either of two BMP inhibitors reduced hepatic triglyceride content in diabetic (db/db) mice. BMP inhibitor-induced decrease in hepatic triglyceride levels was associated with decreased mRNA encoding Dgat2, an enzyme integral to triglyceride synthesis. Treatment of hepatoma cells with BMP2 induced DGAT2 expression and activity via intracellular SMAD signaling. In humans we identified a rare missense single nucleotide polymorphism in the BMP type 1 receptor ALK6 (rs34970181;R371Q) associated with a 2.1-fold increase in the prevalence of NAFLD. In vitro analyses revealed R371Q:ALK6 is a previously unknown constitutively active receptor. These data show that BMP signaling is an important determinant of NAFLD in a murine model and is associated with NAFLD in humans.

Identification of eQTLs associated with lipid metabolism in Longissimus dorsi muscle of pigs with different genetic backgrounds

Intramuscular fat content and its fatty acid composition affect porcine meat quality and its nutritional value. The present work aimed to identify genomic variants regulating the expression in the porcine muscle (Longissimus dorsi) of 45 candidate genes for lipid metabolism and fatty acid composition in three experimental backcrosses based on the Iberian breed. Expression genome-wide association studies (eGWAS) were performed between the muscle gene expression values, measured by real-time quantitative PCR, and the genotypes of 38,426 SNPs distributed along all chromosomes. The eGWAS identified 186 eSNPs located in ten Sus scrofa regions and associated with the expression of ACSM5, ACSS2, ATF3, DGAT2, FOS and IGF2 (FDR < 0.05) genes. Two expression quantitative trait loci (eQTLs) for IGF2 and ACSM5 were classified as cis-acting eQTLs, suggesting a mutation in the same gene affecting its expression. Conversely, ten eQTLs showed trans-regulatory effects on gene expression. When the eGWAS was performed for each backcross independently, only three common trans-eQTL regions were observed, indicating different regulatory mechanisms or allelic frequencies among the breeds. In addition, hotspot regions regulating the expression of several genes were detected. Our results provide new data to better understand the functional regulatory mechanisms of lipid metabolism genes in muscle.

Effects of waterborne exposure to 17β-estradiol on hepatic lipid metabolism genes in tilapia (Oreochromis niloticus)

Estradiol is a steroid hormone that regulates multiple physiological processes in teleosts. The present study aimed to investigate the role of 17β-estradiol (E2) in lipid metabolism in the liver of Nile tilapia. Waterborne exposures were performed using different concentrations of E2 (0 ng/L, 10 ng/L, 25 ng/L and 50 ng/L) and its receptor inhibitor ICI 182,780 (ICI) (1 μM/L). Subsequently, the survival rate and hepatosomatic index (HSI) of tilapia were measured and no significant changes could be found following E2 exposure. Gene expression of VLDL assembly, lipoprotein lipase and lipoprotein receptor, fatty acid uptake and triacylglycerol metabolism were detected by qPCR. E2 exposure up-regulated the expression of lipid metabolism-related genes MTP, LPL, CD36 and DGAT2. ICI exposure inhibited the level of LDLR in the liver of tilapia. This showed that E2 stimulated the MTP expression and promoted VLDL synthesis. In addition, E2 waterborne exposure induced secretion of LDLR and LPL and promoted fatty acid production. Moreover, E2 up-regulated the expression of CD36 to accelerate the transport of fatty acids. Overall, our study provides new information for evaluating Environmental estrogens (EEs) effects on farming fishes and endocrinological regulation on lipid metabolism in fish.

MON-600 Hydroethanolic Extract of Lampaya Medicinalis Phil. (Verbenaceae) Decreases Intracellular Triglycerides and Proinflammatory Marker Expression in Fatty Acid-Exposed HepG2 Hepatocytes

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common hepatic chronic disease worldwide. NAFLD is characterized by an abnormal triglyceride (TG) accumulation (steatosis) in the liver, that may lead to hepatic inflammation (1). DGAT2 is a key enzyme that catalyzes the final step of TG synthesis and whose expression is elevated in NAFLD (2). FABP4 is a transporter of intracellular lipids and its levels are related with inflammation, characterized by a high expression of proinflammatory cytokines such as TNF-α, IL-6 and IL-1β. Palmitic acid (PA, C16:0) and oleic acid (OA; C18:1) are two of the most abundant fatty acids that participate in the formation of TGs in hepatic cells in vivo and in vitro (3). Lampaya medicinalis Phil. (Verbenaceae) is a small bush that grows in the “Puna atacameña” in the North of Chile. The infusion from leaves and aerial parts of the plant has been used by local ethnic groups to treat and cure inflammatory diseases (4). The aim of this study was to assess in vitro the effect of the hydroalcoholic extract of Lampaya medicinalis (HEL) against OA/PA-induced steatosis and proinflammatory marker expression in HepG2 hepatocytes. Methods: HEL (0.01, 0.1, 1, 10 μg/mL) cytotoxicity potential (48 h) was evaluated by Trypan blue exclusion. Cells were exposed for 48 h to 1 mM OA/PA (2:1) in the presence or not of 0.01 or 10 μg/mL HEL. Intracellular TGs were assessed with Oil Red O staining and quantified with Nile Red reagent by fluorimetry. mRNA expression of DGAT2, TNF-α, IL-6 and IL-1β was evaluated by qRT-PCR. FABP4 content was assessed by Western blot. The levels of TNF-α and IL-6 in the culture media were analysed by ELISA. Results: HEL was not cytotoxic at any concentration assessed (n=4; p>0.05). The increased content of TG induced by OA/PA was reduced in the presence of HEL (n=7; p<0.05), showing a strong consistency with Oil Red O staining. The increase in the protein content of FABP4 as well as the increment in mRNA expression of DGAT2, TNF-α, IL-6 and IL-1β induced by OA/PA were lower in cells co-exposed to HEL (n=6-9; p<0.05). The incubation with HEL+OA/PA reduced proinflammatory cytokine levels in culture media compared to cells exposed to OA/PA alone (n=6-7; p<0.05). Conclusion: HEL reduces the OA/PA-induced increase in intracellular TG, DGAT2 and proinflammatory cytokine expression and FABP4 content, as well as the levels of secreted proinflammatory cytokines in HepG2 cells. These findings suggest a protective role for HEL against OA/PA-induced steatosis and inflammation, and therefore that Lampaya medicinalis may represent a promising therapeutic tool for pathologies such as NAFLD.

MP51-07 LOSS OF EPHRIN SIGNALING ALTERS THE LIPID RHEOSTAT ASSOCIATED WITH PROSTATE CANCER RACIAL DISPARITIES

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (MP51)1 Apr 2020MP51-07 LOSS OF EPHRIN SIGNALING ALTERS THE LIPID RHEOSTAT ASSOCIATED WITH PROSTATE CANCER RACIAL DISPARITIES Alejandro Morales, Victoria Gil, Susan Crawford, Simon Hayward, and Omar Franco* Alejandro MoralesAlejandro Morales More articles by this author , Victoria GilVictoria Gil More articles by this author , Susan CrawfordSusan Crawford More articles by this author , Simon HaywardSimon Hayward More articles by this author , and Omar Franco*Omar Franco* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000913.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The presence of abdominal central obesity in prostate cancer (PCa) patients is associated with aggressive tumors. Compared to Caucasians (CA), African American (AA) men are at highest risk of developing and dying from PCa. The Center for Disease Control reports racial disparities in adult obesity (38.4% AA vs. 28.6% in CA). In an obese environment, cancer cells store fatty acids in lipid droplets (LD), composed of triacylglycerols (TAG) and cholesterol esters, which can be used as an energy source to sustain proliferation and migration. Decreased gene expression of members of the ephrin family have been linked to PCa-RD. Here we show the functional consequences of aberrant ephrin signaling in lipid metabolism on PCa tumorigenesis in AA compared to CA. METHODS: The density and size of LD were quantified in PCa specimens from different racial backgrounds. EPHB2 signaling was manipulated in PCa cells using siRNA and CRISPR approaches. LD were quantified under basal and obesogenic (added oleic acid) environments. Molecules involved in TAG metabolism were assessed. The effects of EPHB2 loss on PCa cells growth and local invasion was tested in vitro and in vivo. RESULTS: LD density increased in high grade tumors. Under obesogenic conditions, PCa cells from AA showed increased proliferation compared to CA. Loss of EPHB2 increased the tumorigenicity of PCa cells. Quantification of LD profiles showed racial differences. Alteration of EPHB2 led to differential expression of TAG-associated molecules DGAT1, DGAT2 and ATGL in AA compared to CA. Targeting key TAG molecules associated with EPHB2-induced effects reduced the tumorigenicity of PCa cells. Centrosome amplification and microtubule organizing center (MTOC) were associated with lipid metabolic changes in cells with decreased EphB2 signaling. CONCLUSIONS: Loss of EPHB2 signaling in PCa cells triggers aberrant lipid metabolism leading to excessive accumulation of LD. Because loss of ephrin signaling is associated with PCa-RD, this lipid-rich energy status may serve as a source of energy to fuel a more aggressive phenotype observed in AA. Several TAG inhibitors are currently being evaluated in clinical trials as anti-obesity agents. The potential of these drugs to elicit anti-tumorigenic utility in patients with altered ephrin levels requires further study. Source of Funding: R01 CA242920-01 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e766-e766 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alejandro Morales More articles by this author Victoria Gil More articles by this author Susan Crawford More articles by this author Simon Hayward More articles by this author Omar Franco* More articles by this author Expand All Advertisement PDF downloadLoading ...

Effects of dietary methionine and cysteine restriction on plasma biomarkers, serum fibroblast growth factor 21, and adipose tissue gene expression in women with overweight or obesity: a double-blind randomized controlled pilot study

BackgroundDietary restriction of methionine and cysteine is a well-described model that improves metabolic health in rodents. To investigate the translational potential in humans, we evaluated the effects of dietary methionine and cysteine restriction on cardiometabolic risk factors, plasma and urinary amino acid profile, serum fibroblast growth factor 21 (FGF21), and subcutaneous adipose tissue gene expression in women with overweight and obesity in a double-blind randomized controlled pilot study.MethodsTwenty women with overweight or obesity were allocated to a diet low (Met/Cys-low, n = 7), medium (Met/Cys-medium, n = 7) or high (Met/Cys-high, n = 6) in methionine and cysteine for 7 days. The diets differed only by methionine and cysteine content. Blood and urine were collected at day 0, 1, 3 and 7 and subcutaneous adipose tissue biopsies were taken at day 0 and 7.ResultsPlasma methionine and cystathionine and urinary total cysteine decreased, whereas FGF21 increased in the Met/Cys-low vs. Met/Cys-high group. The Met/Cys-low group had increased mRNA expression of lipogenic genes in adipose tissue including DGAT1. When we excluded one participant with high fasting insulin at baseline, the Met/Cys-low group showed increased expression of ACAC, DGAT1, and tendencies for increased expression of FASN and SCD1 compared to the Met/Cys-high group. The participants reported satisfactory compliance and that the diets were moderately easy to follow.ConclusionsOur data suggest that dietary methionine and cysteine restriction may have beneficial effects on circulating biomarkers, including FGF21, and influence subcutaneous adipose tissue gene expression. These results will aid in the design and implementation of future large-scale dietary interventions with methionine and cysteine restriction.Trial registration ClinicalTrials.gov Identifier: NCT03629392, registration date: 14/08/2018 https://clinicaltrials.gov/ct2/show/NCT03629392.

DGAT2 partially compensates for lipid-induced ER stress in human DGAT1-deficient intestinal stem cells

Dietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future.

Western Diet-Induced Metabolic Alterations Affect Circulating Markers of Liver Function before the Development of Steatosis.

Since nutrition might have a significant impact on liver function, we analyzed the early effect of Western-type diet on hepatic tissue and lipid and drug metabolism in Wistar–Kyoto rats (n = 8); eight rats fed with a standard diet were used as controls. Histological analysis of liver tissue was performed, and plasma biochemical parameters were measured. Plasma concentration of six bile acids was determined by ultra-liquid chromatography-tandem mass spectrometry UHPLC-MS/MS. Hepatic gene expressions of enzymes involved in drug and lipid metabolism were assessed by means of real-time reverse transcription (qRT)-PCR. Liver of rats fed with a Western diet did not show macroscopic histological alterations, but number and diameter of lipid droplets increased, as well as DGAT1, GPAT4, SCD, FASN and SREBP2 expression. Furthermore, Western diet-fed animals showed an increase in the activation of hepatic stellate cells and macrophage number in liver tissue, as well as a significant increase in AST and bilirubin levels (p < 0.01), and in the LDL:HDL cholesterol ratio (p < 0.001). Plasma chenodeoxycholic acid concentration increased significantly, whereas cholic acid decreased (p < 0.05), and cytochrome P450 genes were generally downregulated. Significant changes in hepatic lipid and drug metabolism are early induced by the Western diet, prior to steatosis development. Such changes are associated with a peculiar alteration in circulating bile acids, which could represent an early marker of non-alcoholic fatty liver disease (NAFLD) development.

Short-chain Fatty Acids, 4-phenylbutyric Acid and R-α-lipoic Acid, Downregulate mTORC1-dependent Intestinal Fat Absorption (P08-115-19)

ObjectivesKnowing that (i) high-fat diets upregulate inflammatory biomarkers in the small intestine and activate the mechanistic target of rapamycin complex 1 (mTORC1, a conserved protein kinase) and (ii) mTORC1 drives hepatic VLDL secretion, we investigated the role of intestinal mTORC1 in fat absorption and the lipid-regulating properties of 4-phenylbutyric acid (PBA) and R-α-lipoic acid (LA) in relation to mTORC1. MethodsLentivirus shRNA-based gene editing was utilized to create three stable Caco-2 cell lines that exhibit low, normal or high mTORC1 activity. Fat absorption was investigated in differentiated stable Caco-2 cells grown with oleate/albumin in Transwell permeable inserts. ResultsGenetic ablation of mTORC1 revealed that mTORC1 is required for the production and secretion of apoB-containing chylomicrons in Caco-2 cells. Rapamycin, an inhibitor of mTORC1, confirmed this finding as it lowered the secretion of apoB48 (−75%) and apoB100 (−47%) (P < 0.05). mTORC1 deletion was accompanied by the downregulation of APOB (−87%), MTTP (−94%), FASN (−76%), DGAT1 (−77%) and DGAT2 (−81%) genes (all P < 0.05); lower cellular abundance of apoB100 (−89%), MTTP (−63%) and FASN (−80%) (all P < 0.05); and lower media abundance of apoB48 (−66%), apoB100 (−51%) and triacylglycerols (−69%) (all P < 0.05). In Caco-2 cells harboring high mTORC1 activity, PBA or LA decreased the amounts of secreted apoB48 (−58% or −22%, respectively), apoB100 (−54% or −43%), and triacylglycerols (−46% or −38%), and the gene expression of APOB (−48% or −33%), MTTP (−54% or −31%), SREBF1 (−39% or −37%), FASN (−84% or −38%), and DGAT2 (−76% or −47%) (all P < 0.05). ConclusionsA functional intestinal mTORC1 is indispensable for chylomicron production. Intake of short-chain fatty acid, PBA or LA, may be useful to prevent chylomicron overproduction-associated hypertriglyceridemia and postprandial inflammation. Funding SourcesUSDA Hatch Act, USDA-AFRI.

Maternal heat stress regulates the early fat deposition partly through modification of m6A RNA methylation in neonatal piglets.

It is known that heat stress induces various physiological challenges in livestock production including changes in lipid metabolism. However, the molecular mechanism of how heat stress regulates lipid metabolism at the mRNA level is still largely unknown. N6-methyl-adenosine (m6A) is the most common and abundant modification on RNA molecules present in eukaryotes, which affects almost all aspects of RNA metabolism and thus gives us the hint that it may participate in changes of gene expression of lipid metabolism during heat stress. Therefore, the purpose of the present study was to investigate the effect of heat stress on fat metabolism in 21-day Large White × Landrace piglets from sows challenged by heat stress from day 85 of gestation until day 21 of lactation. We measured the expression of heat shock proteins (HSPs), genes associated with lipid metabolism, m6A-related enzymes, and m6A levels in abdominal fat and liver of offspring piglets. Our results showed that high ambient temperature significantly increased the expression of HSP70 (P < 0.01) in both liver and abdominal fat and upregulated HSP27 in the liver (P < 0.05). Additionally, genes involved in fat metabolism such as ACACA, FASN, DGAT1, PPAR-γ, SREBP-1c, and FABP4 were upregulated in abdominal fat in the experimental group challenged by high ambient temperature. In the liver, heat stress increased the mRNA expression of DGAT1, SREBP-1c, and CD36 and decreased ATGL and CPT1A expression (P < 0.05). The m6A level was higher in the heat stress group compared with the control group in the liver and abdominal fat of offspring piglets (P < 0.01). Notably, heat stress also increased gene expression of METTL14, WTAP, FTO, and YTHDF2 (P < 0.05) in both abdominal fat and liver. The protein abundances of METTL3, METTL14, and FTO were upregulated after heat stress in abdominal fat (P < 0.05) but not in the liver. Although there was no difference in the protein abundance of YTHDF2 in abdominal fat, its level was increased in the liver (P < 0.05). In conclusion, our findings showed that heat stress increased expression of genes involved in lipogenesis, which provided scientific evidence to the observation of increased fatness in pigs under heat stress. We also demonstrated a possible mechanism that m6A RNA modification may be associated with these changes in lipid metabolism upon heat stress.