Adipocyte autophagy limits gut inflammation by controlling oxylipin and IL-10.

Lipids play a major role in inflammatory diseases by altering inflammatory cell functions, either through their function as energy substrates or as lipid mediators such as oxylipins. Autophagy, a lysosomal degradation pathway that limits inflammation, is known to impact on lipid availability, however, whether this controls inflammation remains unexplored. We found that upon intestinal inflammation visceral adipocytes upregulate autophagy and that adipocyte-specific loss of the autophagy gene Atg7 exacerbates inflammation. While autophagy decreased lipolytic release of free fatty acids, loss of the major lipolytic enzyme Pnpla2/Atgl in adipocytes did not alter intestinal inflammation, ruling out free fatty acids as anti-inflammatory energy substrates. Instead, Atg7-deficient adipose tissues exhibited an oxylipin imbalance, driven through an NRF2-mediated upregulation of Ephx1. This shift reduced secretion of IL-10 from adipose tissues, which was dependent on the cytochrome P450-EPHX pathway, and lowered circulating levels of IL-10 to exacerbate intestinal inflammation. These results suggest an underappreciated fat-gut crosstalk through an autophagy-dependent regulation of anti-inflammatory oxylipins via the cytochrome P450-EPHX pathway, indicating a protective effect of adipose tissues for distant inflammation.

Obesity-induced changes in cancer cells and their microenvironment: Mechanisms and therapeutic perspectives to manage dysregulated lipid metabolism.

Obesity has been closely related to cancer progression, recurrence, metastasis, and treatment resistance. We aim to review recent progress in the knowledge on the obese macroenvironment and the generated adipose tumor microenvironment (TME) inducing lipid metabolic dysregulation and their influence on carcinogenic processes. Visceral white adipose tissue expansion during obesity exerts systemic or macroenvironmental effects on tumor initiation, growth, and invasion by promoting inflammation, hyperinsulinemia, growth-factor release, and dyslipidemia. The dynamic relationship between cancer and stromal cells of the obese adipose TME is critical for cancer cell survival and proliferation as well. Experimental evidence shows that secreted paracrine signals from cancer cells can induce lipolysis in cancer-associated adipocytes, causing them to release free fatty acids and acquire a fibroblast-like phenotype. Such adipocyte delipidation and phenotypic change is accompanied by an increased secretion of cytokines by cancer-associated adipocytes and tumor-associated macrophages in the TME. Mechanistically, the availability of adipose TME free fatty acids and tumorigenic cytokines concomitant with the activation of angiogenic processes creates an environment that favors a shift in the cancer cells toward an aggressive phenotype associated with increased invasiveness. We conclude that restoring the aberrant metabolic alterations in the host macroenvironment and in adipose TME of obese subjects would be a therapeutic option to prevent cancer development. Several dietary, lipid-based, and oral antidiabetic pharmacological therapies could potentially prevent tumorigenic processes associated with the dysregulated lipid metabolism closely linked to obesity.

Nuciferine protects bovine hepatocytes against free fatty acid-induced oxidative damage by activating the transcription factor EB/peroxisome proliferator-activated receptor γ coactivator 1 alpha pathway.

Excessive free fatty acid (FFA) oxidation and related metabolism are the major cause of oxidative stress and liver injury in dairy cows during the early postpartum period. In nonruminants, activation of transcription factor EB (TFEB) can improve cell damage and reduce the overproduction of mitochondrial reactive oxygen species. As a downstream target of TFEB, peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α, gene name PPARGC1A) is a critical regulator of oxidative metabolism. Nuciferine (Nuc), a major bioactive compound isolated from the lotus leaf, has been reported to possess hepatoprotective activity. Therefore, the objective of this study was to investigate whether Nuc could protect bovine hepatocytes from FFA-induced lipotoxicity and the underlying mechanisms. A mixture of FFA was diluted in RPMI-1640 basic medium containing 2% low fatty acid bovine serum albumin to treat hepatocytes. Bovine hepatocytes were isolated from newborn calves and treated with various concentrations of FFA mixture (0, 0.3, 0.6, or 1.2 mM) or Nuc (0, 25, 50, or 100 µM), as well as co-treated with 1.2 mM FFA and different concentrations of Nuc. For the experiments of gene silencing, bovine hepatocytes were transfected with small interfering RNA targeted against TFEB or PPARGC1A for 36 h followed by treatment with 1.2 mM FFA for 12 h in presence or absence of 100 µΜ Nuc. The results revealed that FFA treatment decreased protein abundance of nuclear TFEB, cytosolic TFEB, total (t)-TFEB, lysosome-associated membrane protein 1 (LAMP1) and PGC-1α and mRNA abundance of LAMP1, but increased phosphorylated (p)-TFEB. In addition, FFA treatment increased the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and decreased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in bovine hepatocytes. Moreover, FFA administration enhanced the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactose dehydrogenase (LDH) in the medium of FFA-treated hepatocytes, but reduced the content of urea. In FFA-treated bovine hepatocytes, Nuc administration increased TFEB nuclear localization and the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, decreased the contents of MDA and H2O2 and the protein abundance of p-TFEB, and enhanced the activities of CAT and GSH-Px in a dose-dependent manner. Consistently, Nuc administration reduced the activities of ALT, AST, and LDH and increased the content of urea in the medium of FFA-treated hepatocytes. Importantly, knockdown of TFEB reduced the protein abundance of p-TFEB, t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, and impeded the beneficial effects of Nuc on FFA-induced oxidative damage in bovine hepatocytes. In addition, PPARGC1A silencing did not alter Nuc-induced nuclear translocation of TFEB, increase of the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, or decrease of the protein abundance of p-TFEB, whereas it partially reduced the beneficial effects of Nuc on FFA-caused oxidative injury. Taken together, Nuc exerts protective effects against FFA-induced oxidative damage in bovine hepatocytes through activation of the TFEB/PGC-1α signaling pathway.

Using underwater currents as an occupational enrichment method to improve the stress status in rainbow trout.

This study investigated the effects of occupational enrichment, specifically underwater currents, on the stress status of rainbow trout (Oncorhynchus mykiss). A total of 540 fish were divided into three groups: control tanks without artificial currents (CO), tanks with randomly fired underwater currents (RFC), and tanks with continuous current throughout the day (CT). After 30 days, half of the fish in each group underwent a 5-day pre-slaughter fasting (5D), while the others were fed until the day before slaughter (0D). Fish in the RFC group exhibited lower levels of plasma cortisol and acetylcholinesterase enzyme activity in hypothalamus and optic tract than other groups, suggesting an improved stress status. RFC group also showed higher levels of non-esterified fatty acids (NEFA) in 5D fish and higher liver glycogen stores, suggesting improved energy reserves. In comparison, the CT group had higher LDH levels, possibly due to their increased swimming activity. The CO group had significantly lower NEFA levels at 5D compared to the RFC group, suggesting lower energy reserves. The RFC fish had darker and yellow-reddish skin and liver color, suggesting an improved stress status and lower lipid reserves, respectively. Overall, although a significant stress response was not observed in fasted individuals, possibly due to the relatively short fasting period, the study suggests that providing occupational enrichment using randomly fired underwater currents for 1 month helped to improve stress status in rainbow trout, indicating that occupational enrichment during the grow-out phase can positively impact the welfare of rainbow trout during routine handling procedures.

Inositol polyphosphate multikinase modulates free fatty acids-induced insulin resistance in primary mouse hepatocytes.

Insulin resistance is a critical mediator of the development of nonalcoholic fatty liver disease (NAFLD). An excess influx of fatty acids to the liver is thought to be a pathogenic cause of insulin resistance and the development of NAFLD. Although elevated levels of free fatty acids (FFA) in plasma contribute to inducing insulin resistance and NAFLD, the molecular mechanism is not completely understood. This study aimed to determine whether inositol polyphosphate multikinase (IPMK), a regulator of insulin signaling, plays any role in FFA-induced insulin resistance in primary hepatocytes. Here, we show that excess FFA decreased IPMK expression, and blockade of IPMK decrease attenuated the FFA-induced suppression of protein kinase B (Akt) phosphorylation in primary mouse hepatocytes (PMH). Moreover, overexpression of IPMK prevented the FFA-induced suppression of Akt phosphorylation by insulin, while knockout of IPMK exacerbated insulin resistance in PMH. In addition, treatment with MG132, a proteasomal inhibitor, inhibits FFA-induced decrease in IPMK expression and Akt phosphorylation in PMH. Furthermore, treatment with the antioxidant N-acetyl cysteine (NAC) significantly attenuated the FFA-induced reduction of IPMK and restored FFA-induced insulin resistance in PMH. In conclusion, our findings suggest that excess FFA reduces IPMK expression and contributes to the FFA-induced decrease in Akt phosphorylation in PMH, leading to insulin resistance. Our study highlights IPMK as a potential therapeutic target for preventing insulin resistance and NAFLD.

Identification of newly synthetized proteins by mass spectrometry to understand palmitate-induced early cellular changes in pancreatic islets.

Obesity and lipid metabolism dysregulation are often associated with insulin resistance, and can lead to type 2 diabetes. However, mechanisms linking insulin resistance, high levels of plasma free fatty acids (FFA), and ß cell failure remain unclear. The aim of this work was to search for proteins whose synthesis was modified by a short exposure to FFA. This could help in the future to identify molecular mechanisms underlying islet dysfunction in the presence of FFA. Therefore, we assessed by mass spectrometry de novo protein synthesis of freshly isolated rat islets after palmitate short exposure. Quantitative proteome and secretome analyses were performed by combining metabolic incorporation of azidohomoalanine (AHA) and pulse labeling with stable isotope labeling by amino acids in cell culture (SILAC). We showed that pancreatic islets, in response to 4-h exposure to palmitate, increased the synthesis of ribosomal proteins and proteins of the cytoskeleton, and increased their secretion of proteins involved in insulin synthesis and insulin secretion, as well as insulin itself. First, these results show that de novo protein quantification analysis by LC-MS/MS is a useful method to investigate cellular modifications induced by FFA on pancreatic islets. Also, these results show that short exposure to palmitate increases the expression of ribosomal proteins and proteins involved in insulin secretion, and it remains to be determined if these effects are responsible or linked to the harmful effect of palmitate on ß cells.NEW & NOTEWORTHY These results show that pancreatic rat islets cultured with palmitate mainly increase synthesis of ribosomal proteins and some proteins of the cytoskeleton. They also show a significant increase of secreted proteins involved in insulin synthesis and insulin secretion, as well as insulin itself. These data provide information to understand the mechanisms of ß cell failure induced by lipotoxicity via the identification of all newly synthesized proteins in islets in response to short-term exposure to palmitate.

Effects of myonectin on porcine intramuscular adipocyte differentiation and exogenous free fatty acid utilization.

As an important factor secreted by skeletal muscle, myonectin can regulate lipid metabolism and energy metabolism, but its role in the utilization of peripheral free fatty acids (FFAs) by porcine intramuscular fat cells remains to be further investigated. In this study, porcine intramuscular adipocytes were treated with recombinant myonectin and palmitic acid (PA), either alone or in combination, and then were examined for their uptake of exogenous FFAs, intracellular lipid synthesis and catabolism, and mitochondrial oxidation of fatty acids. The results showed that myonectin decreased the area of lipid droplets in intramuscular adipocytes (p < 0.05) and significantly increased (p < 0.05) the expression levels of hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL). Moreover, myonectin can up-regulate the expression of p38 mitogen-activated protein kinase (p38 MAPK). Myonectin significantly promoted the uptake of peripheral FFAs (p < 0.01), improved (p < 0.05) the expression of fatty transport protein 1 (FATP1) and fatty acid binding protein 4 (FABP4) in intramuscular adipocytes. Myonectin also significantly increased (p < 0.05) the expression levels of fatty acid oxidation markers: transcription factor (TFAM), uncoupling protein-2 (UCP2) and oxidative respiratory chain marker protein complex I (NADH-CoQ) in mitochondria of intramuscular adipocytes. In summary, myonectin promoted the absorption, transport, and oxidative metabolism of exogenous FFAs in mitochondria, thereby inhibiting lipid deposition in porcine intramuscular adipocytes.

Free Fatty Acids Induce Lipid Accumulation, Autophagy, and Apoptosis in Human Sebocytes.

BACKGROUND: A disruption of sebocyte differentiation and lipogenesis has fatal consequences and can cause a wide spectrum of skin diseases, from acne vulgaris to sebaceous carcinoma, however, the relevant molecular mechanisms have not been fully clarified. OBJECTIVES: The induction of autophagy and apoptosis in human sebocytes in response to biologically relevant fatty acids was investigated. METHODS: Free fatty acids (arachidonic acid, linoleic acid, palmitic acid, and palmitoleic acid) and the pan-caspase inhibitor QVD-Oph were added to the supernatant of cultured human SZ95 sebocytes. Individual relevant proteins were analyzed by Western blotting. Apoptosis and cell viability were determined, and typical autophagy structures were detected through electron microscopy. To obtain cell growth curves, cell confluence was continuously monitored by real-time cell analysis. RESULTS: Fatty acids induced the development of intracellular lipid droplets with subsequent apoptosis, whereas arachidonic acid caused the most rapid effect. Cleavage products of caspase-3 were only detected in arachidonic acid-induced apoptosis. The high basal apoptotic rate of cultured SZ95 sebocytes was strongly suppressed by QVD-Oph. Fatty acid-induced apoptosis was also markedly inhibited by QVD-Oph, whereas intracellular lipid droplets further accumulated. While cell viability after incubation with linoleic acid, palmitic acid, or palmitoleic acid and QVD-Oph was comparable with that of non-treated controls, arachidonic acid significantly reduced cell viability and cell density despite the concomitant pan-caspase inhibitor treatment. Using electron microscopy, typical autophagy structures were detected, such as autophagosomes and autolysosomes, at the basal level, which became more pronounced after treatment with fatty acids. CONCLUSIONS: Our findings contribute to a better understanding of the inflammation-associated mechanisms of lipogenesis and cell death induction in human sebocytes and may help to unveil the effects of fatty acid-rich human nutrition.

Exposure to Chlorpyrifos Alters Proliferation, Differentiation and Fatty Acid Uptake in 3T3-L1 Cells.

Organophosphorus pesticides (OPs) are important factors in the etiology of many diseases, including obesity and type 2 diabetes mellitus. The aim of this study was to investigate the effect of a representative of OPs, chlorpyrifos (CPF), on viability, proliferation, differentiation, and fatty acid uptake in 3T3-L1 cells. The effect of CPF exposure on preadipocyte proliferation was examined by the MTT, NR, and BrdU assays. The impact of CPF exposure on the differentiation of preadipocytes into mature adipocytes was evaluated by Oil Red O staining and RT-qPCR. The effect of CPF on free fatty acid uptake in adipocytes was assessed with the fluorescent dye BODIPY. Our experiments demonstrated that exposure to CPF decreased the viability of 3T3-L1 cells; however, it was increased when the cells were exposed to low concentrations of the pesticide. Exposure to CPF inhibited the proliferation and differentiation of 3T3-L1 preadipocytes. CPF exposure resulted in decreased lipid accumulation, accompanied by down-regulation of the two key transcription factors in adipogenesis: C/EBPα and PPARγ. Exposure to CPF increased basal free fatty acid uptake in fully differentiated adipocytes but decreased this uptake when CPF was added during the differentiation process. Increased free fatty acid accumulation in fully differentiated adipocytes may suggest that CPF leads to adipocyte hypertrophy, one of the mechanisms leading to obesity, particularly in adults. It can therefore be concluded that CPF may disturb the activity of preadipocytes and adipocytes, although the role of this pesticide in the development of obesity requires further research.

Differential effects of single fatty acids and fatty acid mixtures on the phosphoinositide 3-kinase/Akt/eNOS pathway in endothelial cells.

SCOPE: Dietary fat composition is an important modulator of vascular function. Non-esterified fatty acids (NEFA) enriched in saturated fatty acids (SFA) are thought to reduce vascular reactivity by attenuating insulin signalling via vasodilator pathways (phosphoinositide 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS)) and enhancing signalling via pro-inflammatory pathways. METHODS: To examine the effects of fatty acids on these pathways, human aortic endothelial cells were incubated with single fatty acids, and mixtures of these fatty acids to mimic typical NEFA composition and concentrations achieved in our previous human study. RNA was extracted to determine gene expression using real-time RT-PCR and cell lysates prepared to assess protein phosphorylation by Western blotting. RESULTS: Oleic acid (OA, 100 µM) was shown to down regulate expression of the insulin receptor, PTEN and a PI3K catalytic (p110ß) and regulatory (p85α) subunit compared to palmitic, linoleic and stearic acids (P < 0.04), and promote greater eNOS phosphorylation at Ser1177. Both concentration and composition of the SFA and SFA plus n-3 polyunsaturated fatty acids (PUFA) mixtures had significant effects on genes involved in the PI3K/Akt pathway. Greater up-regulation was found with 800 than 400 µM concentration (respective of concentrations in insulin resistant and normal individuals), whereas greater down-regulation was evident with SFA plus n-3 PUFA than SFA mixture alone. CONCLUSION: Our findings provide novel insights into the modulation of the PI3K/Akt/eNOS pathway by single fatty acids and fatty acid mixtures. In particular, OA appears to promote signalling via this pathway, with further work required to determine the primary molecular site(s) of action.

The relationship between free fatty acids and mitochondrial oxidative stress damage to trophoblast cell in preeclampsia.

AIM: To investigate the effects of free fatty acids on mitochondrial oxidative stress and the pathogenesis of preeclampsia. METHODS: Human primary trophoblast cells at 6-8 weeks of gestation were retrieved and cultured to 70-80% confluence, then incubated in serum from women with a normal pregnancy (normal pregnancy group), women with preeclampsia (PE group), and a combination of serum from women with 24 h preeclampsia-like symptoms and free fatty acids (FFA group). Mitochondrial membrane potential was assessed by fluorescent dye concurrent with detection of membrane channel conversion pore activity by fluorescence microscope. Enzyme labeling instruments and RT-PCR were used to detect mitochondrial DNA (mtDNA) levels. RESULTS: The preeclampsia and free fatty acids groups both exhibited significantly higher levels of mitochondria oxidative stress damage when compared to the normal pregnancy group. However, no significant differences in mitochondrial oxidative stress damage were observed between the FFA and PE groups. CONCLUSIONS: Serum free fatty acids might play an important role in the pathogenesis of preeclampsia by enhancing mitochondrial oxidative stress damage.

Docosahexaenoic Acid Attenuates the Progression of Nonalcoholic Steatohepatitis by Suppressing the Adipocyte Inflammation via the G Protein-Coupled Receptor 120/Free Fatty Acid Receptor 4 Pathway.

INTRODUCTION: Our previous study demonstrated that docosahexaenoic acid (DHA), an endogenous G protein-coupled receptor 120 (GPR120)/free fatty acid receptor (FFAR) 4 agonist, attenuated the liver inflammation in nonalcoholic steatohepatitis (NASH), while exacerbated liver inflammation was observed in the GPR120/FFAR4 knockout (GPR120/FFAR4KO) mice. Recently, abdominal adiposity has been reported to correlate with the severity of inflammation and fibrosis in patients with NASH. In this study, we investigated whether the activation of GPR120/FFAR4 suppressed the inflammation of the adipose tissue and whether these suppressive effects attenuated the development of NASH. METHODS: A choline-deficient and 0.1% methionine-containing high-fat (CDAHF) diet was used to create a mouse model of NASH. DHA was orally administered to the mice for 1 week. Epididymal fat pads which collected from the control-fed wild-type (WT) or GPR120/FFAR4KO mice were used as ex vivo white adipose tissue (WAT) culture systems. RESULTS: The mice fed a CDAHF diet for 2 weeks showed NASH-like liver diseases. In the WAT of mice fed with the CDAHF diet, inflammation and fibrosis were significantly increased, and the administration of DHA suppressed these phenomena. In an ex vivo adipocyte culture study, DHA dose-dependently suppressed the lipopolysaccharide-induced inflammation in the adipocyte tissue of WT mice, which was reversed by pretreatment with AH7614, a GPR120/FFAR4 antagonist, but not GPR40 or peroxisome proliferator-activated receptor γ antagonist. CONCLUSIONS: These findings suggest that the activation of GPR120/FFAR4 may suppress the inflammation of adipocytes, which could be a key pathway to prevent the development of NASH.

The Beneficial Effects of Triterpenic Acid and Acteoside in an In Vitro Model of Nonalcoholic Steatohepatitis (NASH).

Triterpenic acid (TA) and acteoside (ACT), the major components of APPLIVER and ACTEOS, respectively, have been reported to exert hepatoprotective effects, but the molecular mechanisms remain elusive, particularly in the NAFLD/NASH context. We assessed their effects in our well-established in vitro model resembling the pathophysiological mechanisms involved in NASH. Human hepatocytes and hepatic stellate cells were exposed to free fatty acids (FFA) alone or in combination with APPLIVER and ACTEOS as a mono- or co-culture. Steatosis, inflammation, generation of reactive oxygen species (ROS), and collagen deposition were determined. ACTEOS reduced both the TNF-α and ROS production, and, most importantly, attenuated collagen deposition elicited by the excess of FFA in the co-culture model. APPLIVER also showed inhibition of both TNF-α production and collagen deposition caused by FFA accumulation. The compounds alone did not induce any cellular effects. The present study showed the efficacy of APPLIVER and ACTEOS on pathophysiological mechanisms related to NASH. These in vitro data suggest that these compounds deserve further investigation for possible use in NASH treatment.

Hepatocyte Proteome Alterations Induced by Individual and Combinations of Common Free Fatty Acids.

Non-alcoholic fatty liver disease is a pathology with a hard-to-detect onset and is estimated to be present in a quarter of the adult human population. To improve our understanding of the development of non-alcoholic fatty liver disease, we treated a human hepatoma cell line model, HepG2, with increasing concentrations of common fatty acids, namely myristic, palmitic and oleic acid. To reproduce more physiologically representative conditions, we also included combinations of these fatty acids and monitored the cellular response with an in-depth proteomics approach and imaging techniques. The two saturated fatty acids initially presented a similar phenotype of a dose-dependent decrease in growth rates and impaired lipid droplet formation. Detailed analysis revealed that the drop in the growth rates was due to delayed cell-cycle progression following myristic acid treatment, whereas palmitic acid led to cellular apoptosis. In contrast, oleic acid, as well as saturated fatty acid mixtures with oleic acid, led to a dose-dependent increase in lipid droplet volume without adverse impacts on cell growth. Comparing the effects of harmful single-fatty-acid treatments and the well-tolerated fatty acid mixes on the cellular proteome, we were able to differentiate between fatty-acid-specific cellular responses and likely common lipotoxic denominators.

Physiological Doses of Oleic and Palmitic Acids Protect Human Endothelial Cells from Oxidative Stress.

Oxidative stress has been proposed to be a pathogenic mechanism to induce endothelial dysfunction and the onset of cardiovascular disease. Elevated levels of free fatty acids can cause oxidative stress by increasing mitochondrial uncoupling but, at physiological concentrations, they are essential for cell and tissue function and olive oil free fatty acids have proved to exhibit beneficial effects on risk factors for cardiovascular disease. We hypothesize that realistic concentrations within the physiological range of oleic (OA) and palmitic (PA) acids could be beneficial in the prevention of oxidative stress in vascular endothelium. Hence, pre-treatment and co-treatment with realistic physiological doses of palmitic and oleic acids were tested on cultured endothelial cells submitted to a chemically induced oxidative stress to investigate their potential chemo-protective effect. Cell viability and markers of oxidative status: reactive oxygen species (ROS), reduced glutathione (GSH), malondialdehyde (MDA), glutathione peroxidase (GPx) and glutathione reductase (GR) were evaluated. As a conclusion, the increased ROS generation induced by stress was significantly prevented by a pre- and co-treatment with PA or OA. Moreover, pre- and co-treatment of cells with FFAs recovered the stress-induced MDA concentration to control values and significantly recovered depleted GSH and normalized GPx and GR activities. Finally, pre- and co-treatment of cells with physiological concentrations of PA or OA in the low micromolar range conferred a substantial protection of cell viability against an oxidative insult.

Effects of NEFAs during IVM on pig embryos from granulosa cell-cocultured oocytes.

Circulating levels of nonesterified fatty acids (NEFAs) are elevated in some females, which can impair oocyte maturation and embryo development, and may alter the phenotype of the progeny. However, the effects of NEFAs on human embryo development are not clear due to ethical limitations. Thus, we used pig as the model to investigate the impacts of NEFAs on oocyte and embryo due to their similar reproductive and metabolic physiologies to humans. In this study, porcine cumulus-oocyte complexes were in vitro maturated under a pathologically high concentration of NEFAs (468 µM palmitic acid, 194 µM stearic acid, and 534 µM oleic acid) with the presence of granulosa cell monolayer, in contrast to control without NEFAs. The mature oocytes were fertilized to produce embryos for further analysis of the transcriptome and DNA methylation patterns. The elevated level of NEFAs decreased the blastocyst rate and delayed the blastocyst development. Ingenuity pathway analysis showed that the most affected gene pathways were related mainly to cell activities, metabolism, and inflammation. These findings indicated that oocytes exposed to the exogenous high level of NEFAs during in vitro maturation resulted in altered gene expression and DNA methylation of early embryos, which have detrimental impacts on blastocyst quality.

Leaf Surface Wax Chemicals in Trichosanthes anguina (Cucurbitaceae) Cultivars Mediating Short-Range Attraction and Oviposition in Diaphania indica.

Larval Diaphania indica (Saunders) (Lepidoptera: Crambidae) cause complete defoliation of Trichosanthes anguina L. and reduce crop yield in India. Females lay eggs on the leaf surface, and therefore leaf surface waxes are potentially involved in host selection. Alkanes and free fatty acids are the major constituents of leaf surface waxes, so a study was conducted to determine whether these wax constituents from three T. anguina cultivars (MNSR-1, Baruipur Long, and Polo No.1) could act as short-range attractants and oviposition stimulants in D. indica females. Twenty n-alkanes from n-C14 to n-C36 and 13 free fatty acids from C12:0 to C21:0 were detected in the leaf surface waxes of these cultivars. Heptadecane and stearic acid were predominant among n-alkanes and free fatty acids, respectively, in these cultivars. Females showed attraction towards one leaf equivalent surface wax of each of these cultivars against solvent controls (petroleum ether) in Y-tube olfactometer bioassays. A synthetic blend of heptadecane, eicosane, hexacosane, and stearic acid, a synthetic blend of hexacosane and stearic acid, and a synthetic blend of pentadecane and stearic acid comparable to amounts present in one leaf equivalent surface wax of MNSR-1, Baruipur Long, and Polo No.1, respectively, were short-range attractants and oviposition stimulants in D. indica. Female egg laying responses were similar to each of these blends, providing information that could be used to developing baited traps in integrated pest management (IPM) programs.

Sphingosine-1 Phosphate Lyase Regulates Sensitivity of Pancreatic Beta-Cells to Lipotoxicity.

Elevated levels of free fatty acids (FFAs) have been related to pancreatic beta-cell failure in type 2 diabetes (T2DM), though the underlying mechanisms are not yet fully understood. FFAs have been shown to dysregulate formation of bioactive sphingolipids, such as ceramides and sphingosine-1 phosphate (S1P) in beta-cells. The aim of this study was to analyze the role of sphingosine-1 phosphate lyase (SPL), a key enzyme of the sphingolipid pathway that catalyzes an irreversible degradation of S1P, in the sensitivity of beta-cells to lipotoxicity. To validate the role of SPL in lipotoxicity, we modulated SPL expression in rat INS1E cells and in human EndoC-ßH1 beta-cells. SPL overexpression in INS1E cells (INS1E-SPL), which are characterized by a moderate basal expression level of SPL, resulted in an acceleration of palmitate-mediated cell viability loss, proliferation inhibition and induction of oxidative stress. SPL overexpression affected the mRNA expression of ER stress markers and mitochondrial chaperones. In contrast to control cells, in INS1E-SPL cells no protective effect of oleate was detected. Moreover, Plin2 expression and lipid droplet formation were strongly reduced in OA-treated INS1E-SPL cells. Silencing of SPL in human EndoC-ßH1 beta-cells, which are characterized by a significantly higher SPL expression as compared to rodent beta-cells, resulted in prevention of FFA-mediated caspase-3/7 activation. Our findings indicate that an adequate control of S1P degradation by SPL might be crucially involved in the susceptibility of pancreatic beta-cells to lipotoxicity.

The effects of glucose and fatty acids on CXCL10 expression in skeletal muscle cells.

Skeletal muscles produce secretory factors termed as myokines, which alter physiological functions of target tissues. We recently identified C-X-C chemokine ligand 10 (CXCL10) as a novel myokine, which is downregulated in response to exercise. In the present study, we investigated whether the nutritional changes affect CXCL10 expression in mouse skeletal muscle. Expression of CXCL10 was evaluated in mice fed a normal diet or a high fat diet for 10 weeks. In animals fed on HFD, Cxcl10 expression was significantly induced in fast-twitched muscles, and was accompanied by increased blood glucose and free fatty acid levels. In vitro experiments using C2C12 myotubes suggested that the increased levels of glucose and palmitic acids directly enhanced CXCL10 expression. Interestingly, the effect of palmitic acids was attenuated by palmitoleic acids. Considering its potent angiostatic activity, induction of CXCL10 by nutritional changes may contribute to the impairment of microvascular networks in skeletal muscles.

Effect and mechanism of ginsenoside Rg1-regulating hepatic steatosis in HepG2 cells induced by free fatty acid.

Ginsenoside Rg1 (G-Rg1) is a bioactive phytochemical that has been found to be beneficial for the treatment of several diseases including nonalcoholic fatty liver disease (NAFLD). But there is a lack of literature reporting the effect of G-Rg1 on lipid metabolism balance in NAFLD. We investigated the effect and mechanism of G-Rg1 on lipid metabolism in vitro. We found that G-Rg1 decreased the levels of TG, TC, and MDA, and increased activity of SOD. Results of RT-PCR and western blotting showed that supplementation with G-Rg1 downregulated the expression of PPAR γ, FABP1, FATP2/5, CD36, SREBP1 c, and FASN, while the expression of PPAR ɑ, CPT1, ACOX1, MTTP, and ApoB100 was upregulated, after induction by a free fatty acid. Taken together, we conclude that G-Rg1 inhibits lipid synthesis and lipid uptake, and enhances lipid oxidation and lipid export to reduce hepatic steatosis of HepG2 cells by regulating PPAR ɑ and PPAR γ expression.