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Indole-3-acetic acid alleviates palmitic acid-induced lipid accumulation and endoplasmic reticulum stress via sirtuin 3 in primary bovine hepatocytes

ABSTRACT: Ketotic cows experience severe negative energy balance, in which an increase in circulating non-esterified fatty acids (NEFA), particularly palmitic acid (PA), is a primary cause of liver damage. Although reports indicate that the endogenous tryptophan metabolite indole-3-acetic acid (3-IAA) can protect the liver of non-ruminant animals, its role in mitigating lipotoxicity-induced damage to bovine hepatocytes remains unclear. In this study, ketotic cows showed higher serum β-hydroxybutyrate and NEFA, lower 3-IAA and glucose concentrations, and increased hepatic triacylglycerol (TG) content, which was negatively correlated with serum 3-IAA. In primary bovine hepatocytes, treatment with 200 μmol/L 3-IAA improved cell viability, reduced cytotoxicity, and alleviated PA-induced lipid metabolic disorders and endoplasmic reticulum stress (ERS). RNA sequencing indicated that 3-IAA regulated pathways related to lipid metabolism and ERS. Mechanistically, 3-IAA attenuated PA-induced lipid accumulation, downregulated sterol regulatory element-binding protein 1 (SREBP-1) protein and acetyl-CoA carboxylase α (ACACA) or fatty acid synthase (FASN) mRNA, and concurrently increased peroxisome proliferator-activated receptor α (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) protein abundance and CPT1A mRNA, suggesting reduced lipogenesis and enhanced fatty acid oxidation. In parallel, 3-IAA alleviated ER swelling and suppressed ERS at both the protein and gene levels by reducing the protein abundance of phosphorylated protein kinase R-like endoplasmic reticulum kinase (P-PERK), phosphorylated inositol-requiring enzyme 1α (P-IRE1α), and cleaved activating transcription factor 6 (ATF6) with gene expression of glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and spliced X-box binding protein 1 (sXBP1). Notably, 3-IAA increased sirtuin 3 (SIRT3) mRNA and protein expression in PA-treated hepatocytes, whereas SIRT3 knockdown partially abolished its protective effects. Collectively, these findings indicate that 3-IAA mitigates PA-induced lipid accumulation and ERS via a SIRT3-dependent pathway in bovine hepatocytes, and suggest that 3-IAA may act as an endogenous regulator of hepatic homeostasis in ketotic cows.

ORGANISM(S): Bos taurus

PROVIDER: GSE329246 | GEO | 2026/07/01

REPOSITORIES: GEO

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Project description:Metabolic challenges experienced by dairy cows during the transition between pregnancy and lactation (also known as peripartum), are of considerable interest from a nutrigenomic perspective. The mobilization of large amounts of non-esterified fatty acids (NEFA) leads to an increase in NEFA uptake in the liver, the excess of which can cause hepatic accumulation of lipids and ultimately fatty liver. Interestingly, peripartum NEFA activate the peroxisome proliferator-activated receptor (PPAR), a transcriptional regulator with known nutrigenomic properties. The study of PPAR activation in the liver of periparturient dairy cows is thus crucial; however, current in vitro models of the bovine liver are inadequate, and the isolation of primary hepatocytes is prohibitive and error-prone. The objective of the current study was to evaluate the use of precision-cut liver slices (PCLS) from liver biopsies as a model for PPAR activation in periparturient dairy cows. Four cows were enrolled in the experiment, and PCLS from each were prepared prepartum (-10 DIM) and postpartum (+10 DIM) and treated with a variety of PPAR agonists and antagonist. Gene expression was assayed through RT-qPCR and RNAseq, and intracellular triglyceride concentration was measured. PCLS treated with PPARγ agonist rosiglitazone displayed upregulation of canonical PPAR targets ACADVL and LIPC in the postpartum, while a PPARδ agonists increased expression of PPAR target PDK4; no gene expression changes were detected in the prepartum, and triglyceride concentrations were unchanged between treatments. Transcriptome sequencing revealed considerable differences in response to PPAR agonist, mainly related to pathways involved with lipid metabolism and the immune response. Among differentially expressed genes, a subset of 91 genes were identified as novel putative PPAR targets in the bovine liver, by cross-referencing our results with a publicly-available dataset of predicted PPAR target genes, and supplementing our finding with prior literature. Our results provide important insights on the use of PCLS as a model for assaying PPAR activation in the peripartum.

Project description:Over-conditioning is a risk factor for upregulated pre- and postpartum fat mobilization. Therefore, we hypothesized that over-conditioning at the end of pregnancy leads to the accumulation of lipids in the liver and modifications of the hepatic gene expression pattern. The aim of this study was to evaluate the effect of over- versus normal-conditioning on the hepatic transcriptomic profile of dairy cows at the end of pregnancy. Ten dry multiparous Holstein cows were euthanized two weeks before expected calving. Body condition score (BCS) and backfat thickness (BFT) were evaluated, and blood samples for nonesterified fatty acids (NEFA) were taken before euthanasia. After euthanasia, liver biopsy samples were collected for further assessment of total lipids and RNA sequencing. Five cows were classified as normal-conditioned (BCS = 2.5–3.5) and five as over-conditioned (BCS = 3.75–5). Regression models confirmed that normal-conditioned cows had lower BCS [3.17 ± 0.10 (least square mean ± SE)], BFT (1.29 ± 0.29 cm), and serum NEFA (0.16 ± 0.04 mmol/L) in comparison to over-conditioned cows (4.35 ± 0.21, 3.14 ± 0.43 cm, and 0.38 ± 0.07 mmol/L for BCS, BFT, and NEFA, respectively). Total liver lipid percentage tended to be lower in normal- than over-conditioned cows (4.63 ± 0.40% and 6.06 ± 0.44%, respectively). In comparison to the mean liver lipid percentage of the normal and over conditioned cows, one over-conditioned cow had a relatively low (5.21%) and one normal-conditioned cow had a relatively high (6.07%) liver lipid percentage. Differentially expressed genes (DEGs) analysis (edgeR quasi-likelihood method) showed that normal-conditioned presented two upregulated and five downregulated genes in comparison to over-conditioned cows. Linear discriminant analysis effects size revealed 23 DEG between normal- versus over-conditioned cows. Notably, the liver of normal-conditioned cows had upregulated genes associated with liver functionality (albumin, selenoprotein P, and insulin-like growth factor binding protein 1 and 2). On the other hand, over-conditioned cows had upregulated genes associated with the acute phase response (complement C3, hemopexin, and lipopolysaccharide-binding protein). High basal lipolysis in over-conditioned cows at the end of pregnancy increases liver lipid content and alters the hepatic gene expression pattern to a pro-inflammatory state.

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Project description:Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, and more specifically DNA methylation, in the offspring. Final oocyte maturation and early embryo development coincide with specific methylation changes and both time periods are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect the establishment and maintenance of DNA methylation patterns in oocytes and embryos and subsequently alter transcriptomic profiles and developmental competence of the resultant blastocysts. To do this, bovine oocytes and embryos were exposed to different NEFA concentrations in two separate experiments. In the first experiment, oocytes were matured in vitro for 24 hours in medium containing: 1) physiological (âBASALâ) concentrations of oleic (OA), palmitic (PA) and stearic (SA) or 2) elevated (pathophysiological or âHIGH COMBIâ) concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. The developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray techniques. Data regarding DNA methylation were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to their BASAL-exposed counterparts (19.3 % compared to 23.2 % and 18.2 % compared to 25.3 %, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation profiles and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to an adverse environment. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later stages of life observed in offspring from mothers displaying lipolytic disorders. The effect of elevated NEFA exposure during embryo culture (6.5 days) on gene expression was evaluated by exposing embryos to the BASAL or HIGH COMBI conditions. A total of 1412 oocytes were used, equally distributed between treatments in 4 replicates. Resultant day 7.5 blastocysts were snap frozen for subsequent transcriptome analysis (80 blastocysts in each experiments, equally collected between treatments in 4 replicates).

Project description:Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, and more specifically DNA methylation, in the offspring. Final oocyte maturation and early embryo development coincide with specific methylation changes and both time periods are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect the establishment and maintenance of DNA methylation patterns in oocytes and embryos and subsequently alter transcriptomic profiles and developmental competence of the resultant blastocysts. To do this, bovine oocytes and embryos were exposed to different NEFA concentrations in two separate experiments. In the first experiment, oocytes were matured in vitro for 24 hours in medium containing: 1) physiological (âBASALâ) concentrations of oleic (OA), palmitic (PA) and stearic (SA) or 2) elevated (pathophysiological or âHIGH COMBIâ) concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. The developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray techniques. Data regarding DNA methylation were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to their BASAL-exposed counterparts (19.3 % compared to 23.2 % and 18.2 % compared to 25.3 %, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation profiles and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to an adverse environment. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later stages of life observed in offspring from mothers displaying lipolytic disorders. Two experiments were set up to characterize the epigenomic profile of the resultant day 7.5 blastocysts. In the first (in vitro maturation or IVM) experiment, the effect of elevated NEFA exposure during oocyte maturation (24 h) on DNA methylation profiles was evaluated by exposing oocytes to the BASAL or HIGH COMBI conditions. In the second (in vitro culture or IVC) experiment, the effect of elevated NEFA exposure during embryo culture (6.5 days) on DNA methylation marks was evaluated by exposing embryos to the BASAL or HIGH COMBI conditions. A total of 1039 and 1412 oocytes were used in the IVM and IVC experiment, respectively, equally distributed between treatments in 4 replicates. Resultant day 7.5 blastocysts were snap frozen for subsequent DNA methylation analysis (80 blastocysts in each experiments, equally collected between treatments in 4 replicates).

Project description:Adipose tissue dysfunction results in hepatic lipid overload, driving the progression of liver steatosis and non-alcoholic steatohepatitis. The involvement of adipose tissue-derived small extracellular vesicles (sEVs) in initiating and propagating nonalcoholic fatty liver disease (NAFLD) remains unexplored. Herein, we unveiled that transplanting adipose tissue from high-fat diet (HFD)-fed mice exacerbated insulin resistance and liver steatosis in lean mice, and Sirt3 expression was markedly reduced in adipose tissue from both NAFLD patients and murine NAFLD models. Interestingly, fat-specific Sirt3 overexpression (Sirt3AKI) mice enhanced insulin sensitivity and improved liver steatosis under HFD feeding. Furthermore, adipose tissue-derived sEVs were found to regulate hepatic lipid metabolism in Sirt3AKI mice. In vitro studies demonstrated that Sirt3 overexpressing adipocytes (Sirt3OE) released sEVs to ameliorate lipid accumulation in palmitic acid/oleic acid (P/O)-stimulated hepatocytes. Conversely, sEVs derived from NAFLD patients exacerbated hepatic lipid accumulation in HFD-fed Sirt3AKI mice. MicroRNA sequencing further identified a significant decrease in miR-30a-3p levels in sEVs derived from Sirt3OE adipocytes. Fat-specific miR-30a-3p overexpression worsened hepatic steatosis in HFD-fed Sirt3AKI mice. In contrast, engineered sEVs loaded with miR-30a-3p antagonist or miR-30a-3p inhibition robustly enhanced insulin sensitivity and mitigated hepatic accumulation in HFD-fed mice. Mechanistically, CHIP-qPCR and CHIP-sequencing analyses revealed that Sirt3 suppressed miR-30a-3p transcription through H3K56 acetylation. Dual-luciferase reporter assays validated Beclin1 as a bona fide target mRNA of miR-30a-3p. Taken together, these findings highlight the critical role of exosomal microRNAs in mediating adipocyte-hepatocyte crosstalk, provide novel insights into the mechanisms underlying lipotoxicity in hepatocytes, and suggest miR-30a-3p inhibitor could be a therapeutic agent for NAFLD.

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Indole-3-acetic acid alleviates palmitic acid-induced lipid accumulation and endoplasmic reticulum stress via sirtuin 3 in primary bovine hepatocytes

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