Elevated Fatty Acid Levels Research Articles

1674-P: Stearate Potentiates MIP-1a Production with TNF-a via TLR4/TBK1/IKKe /IRF3 Signal Axis: Significance for Insulin Resistance

Increased levels of plasma free fatty acids (FAs) are an important contributor to chronic inflammation, insulin resistance and other obesity-related complications. However, the molecular mechanisms by which individual FAs contribute to obesity-associated inflammation and disorders remain incompletely understood. Here we report that dietary saturated FAs specifically stearate led to a profound production of macrophage inflammatory protein (MIP)-1α (CCL3) in the presence of TNF-α in monocytes, macrophages and adipose tissues. Using pharmacologic and genetic approaches, we identified the involvement of TLR4/TBK1/IKKε/IRF3 signal axis in this production of MIP-1α in response to stearate and TNF-α. Consistent with this, cultured adipose tissues from TLR4 knockout (KO) mice revealed that stearate and TNF-α treatment did not induce MIP-1α compared with TLR2 KO or wild type (C57BL/6) mice. Moreover, C57BL/6 mice fed a high fat diet (HFD) for 16 weeks showed elevated levels of plasma fatty acids, TNF-α and MIP-1α along with insulin resistance. MIP-1α was significantly correlated with TNF-α. Our human data shows that expression of TLR4/p-IRF3 was elevated in the PBMCs of obese individuals as compared to lean. Furthermore, elevated MIP-1α expression levels in obese fat tissues were significantly correlated with TNF-α and macrophage markers. In conclusion, these findings provide a mechanistic link between stearate and TNF-α for the overproduction of obesity associated MIP-1α, which could be used as a new target for the treatment of obesity-related chronic inflammation/insulin resistance. Disclosure S.P. Kochumon: None. F. Almulla: None. Funding Kuwait Foundation for the Advancement of Science (RA-AH-2016-007)

Oleic acid stimulates monoamine efflux through PPAR-α: Differential effects in diet-induced obesity

Obesity continues to be a growing health concern around the world, and elevated levels of free fatty acids as a result of high-fat intake might play a role in neuroendocrine alterations leading to obesity. However, it is unclear how fatty acids affect neuroendocrine functions and energy metabolism. Since hypothalamic monoamines play a crucial role in regulating neuroendocrine functions relating to energy balance, we investigated the direct effects of oleic acid on hypothalamic monoamines and hypothesized that oleic acid would activate peroxisome proliferator-activated receptor alpha (PPAR-α), a nuclear transcription factor involved with fatty acid metabolism, to affect monoamines. We also hypothesized that this response would be subdued in diet-induced obesity (DIO). To test these hypotheses, hypothalami from Sprague Dawley and DIO rats were incubated with 0 (Control), 0.00132 mM, 0.132 mM, 1.32 mM oleic acid, 50 μM MK 886 (a selective PPAR- α antagonist), or oleic acid + MK 886 in Krebs Ringers Henseleit (KRH) solution. HPLC-EC was used to measure monoamine levels in perfusates. Oleic acid produced a significant increase in norepinephrine, dopamine, and serotonin levels in a dose-dependent manner, and incubation with MK886 blocked these effects. The effect of oleic acid on hypothalamic monoamines was attenuated in DIO rats. These findings suggest that PPARα probably plays an essential role in fatty acid sensing in the hypothalamus, by affecting monoamine efflux and DIO rats are resistant to the effects of oleic acid.

Non-esterified Fatty Acid Induce Dairy Cow Hepatocytes Apoptosis via the Mitochondria-Mediated ROS-JNK/ERK Signaling Pathway.

Elevated plasma non-esterified fatty acid (NEFA) levels and hepatocytes damage are characteristics of ketosis in dairy cows. Oxidative stress is associated with the pathogenesis of NEFA-induced liver damage. However, the exact mechanism by which oxidative stress mediates NEFA-induced hepatocytes apoptosis and liver injury remains poorly understood. The results of the present study demonstrated that NEFA contribute to reactive oxygen species (ROS) generation, resulting in an imbalance between oxidative and antioxidant species, transcriptional activation of p53, transcriptional inhibition of nuclear factor E2-related factor 2 (Nrf2), loss of mitochondria membrane potential (MMP) and release of apoptosis-inducing factor (AIF) and cytochrome c (cyt c) into the cytosol, leading to hepatocytes apoptosis. Besides, NEFA triggered apoptosis in dairy cow hepatocytes via the regulation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), Bcl-2-associated X protein (Bax), B-cell lymphoma gene 2 (Bcl-2), caspase 9 and poly (ADP-ribose) polymerase (PARP). Pretreatment with the inhibitor SP600125 or PD98059 or the antioxidant N-acetylcysteine (NAC) revealed that NEFA-ROS-JNK/ERK-mediated mitochondrial signaling pathway plays a crucial role in NEFA-induced hepatocytes apoptosis. Moreover, the results suggested that the transcription factors p53 and Nrf2 function downstream of this NEFA-ROS-JNK/ERK pathway and are involved in NEFA-induced hepatocytes apoptosis. In conclusion, these findings indicate that the NEFA-ROS-JNK/ERK-mediated mitochondrial pathway plays an important role in NEFA-induced dairy cow hepatocytes apoptosis and strongly suggests that the inhibitors SP600125 and PD98059 and the antioxidant NAC may be developed as therapeutics to prevent hyperlipidemia-induced apoptotic damage in ketotic dairy cows.

Influence of electrochemical advanced oxidation on the long-term operation of an Upflow Anaerobic Sludge Blanket (UASB) reactor treating 4-chlorophenol containing wastewater

Abstract Upflow anaerobic sludge blanket (UASB) systems are of specific interest for the treatment of high organic wastewater since they offer the opportunity to recover energy in the form of methane (biogas). The technology is highly suitable for the degradation of biodegradable organics, but its efficiency can be significantly hampered by the presence of toxic pollutants in the water. In this study, the use of electrochemical advanced oxidation (eAOP) coupled to a lab-scale UASB reactor for the degradation of 4-chlorophenol (4-CP) in wastewater was investigated. The eAOP treatment consisted of the electrochemical production of active chlorine at a Ti/RuO2–IrO2 anode with a fixed current density of 25 mA/cm2 in the recycle of the UASB reactor. It was noticed that the total COD removal efficiency decreased from 75.8% when no 4-CP was present in the wastewater to 25.6% and 13.6% after the addition of 100 ppm and 50 ppm 4-CP, respectively. Furthermore, elevated levels of volatile fatty acids and ORP were observed in the effluent of the reactor, indicating process failure. It was postulated that the decrease in COD removal efficiency was due to the release of high amounts of hypochlorite, causing an oxidative environment in the anaerobic reactor. This hypochlorite may have reacted with several components such as ammonia to form other toxic components (e.g., chloramine).

Hypoxia-Inducible Lipid Droplet-Associated Interacts with DGAT1 to Promote Lipid Storage in Hepatocytes

Lipid droplets (LD) are dynamic organelles that can expand and shrink, driven by fluctuations in the rate of triglyceride synthesis and degradation. Triglyceride synthesis, storage in LD, and degradation are governed by a complex set of LD-associated proteins. One of these LD-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair LD breakdown by inhibiting adipose triglyceride lipase. Here we characterized the physiological role and mechanism of action of HILPDA in hepatocytes. Expression of HILPDA was induced by fatty acids in several hepatoma cell lines. Fluorescence microscopy showed that HILPDA partly colocalizes with LD and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging revealed that HILPDA preferentially localizes to LD that are being remodelled. Deficiency of HILPDA in mouse precision-cut liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labelled fatty acids in LD, respectively, which was independent of adipose triglyceride lipase. Confocal microscopy and Forster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicated that HILPDA colocalizes and physically interacts with DGAT1. In human hepatoma HepG2 cells, HILPDA overexpression increased lipid storage concomitant with an increase in DGAT activity and DGAT1 protein levels. Finally, hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglyceride and plasma alanine aminotransferase levels in mice with non-alcoholic fatty liver disease. Overall, our data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity and DGAT1 protein levels. These findings suggest a novel mechanism in hepatocytes that links elevated fatty acid levels to stimulation of triglyceride synthesis and storage.

Low protein diet and methyl-donor supplements modify testicular physiology in mice.

The link between male diet and sperm quality has received significant investigation. However, the impact diet and dietary supplements have on the testicular environment has been examined to a lesser extent. Here, we establish the impact of a sub-optimal low protein diet (LPD) on testicular morphology, apoptosis and serum fatty acid profiles. Furthermore, we define whether supplementing a LPD with specific methyl donors abrogates any detrimental effects of the LPD. Male C57BL6 mice were fed either a control normal protein diet (NPD; 18% protein; n = 8), an isocaloric LPD (LPD; 9% protein; n = 8) or an LPD supplemented with methyl donors (MD-LPD; choline chloride, betaine, methionine, folic acid, vitamin B12; n = 8) for a minimum of 7 weeks. Analysis of male serum fatty acid profiles by gas chromatography revealed elevated levels of saturated fatty acids and lower levels of mono- and polyunsaturated fatty acids in MD-LPD males when compared to NPD and/or LPD males. Testes of LPD males displayed larger seminiferous tubule cross section area when compared to NPD and MD-LPD males, while MD-LPD tubules displayed a larger luminal area. Furthermore, TUNNEL staining revealed LPD males possessed a reduced number of tubules positive for apoptosis, while gene expression analysis showed MD-LPD testes displayed decreased expression of the pro-apoptotic genes Bax, Csap1 and Fas when compared to NPD males. Finally, testes from MD-LPD males displayed a reduced telomere length but increased telomerase activity. These data reveal the significance of sub-optimal nutrition for paternal metabolic and reproductive physiology.

Flower Infusions From Cornus mas and Cornus kousa Inhibit Aldose Reductase Enzyme, Without Any Effects on Lipotoxicity

Aldose reductase inhibitors are considered to be potential therapeutic agents for chronic diabetic complications. Diabetes mellitus can be accompanied by elevated blood levels of free fatty acids, which can cause lipotoxicity. Herbal extracts and their constituents are promising agents which have the potential for alleviating these complications. Our study was focused on the influence on these effects by flower infusions from Cornus mas L. and Cornus kousa F.Buerger ex Hance. Initially, phenolic compounds were quantified in the dried flowers. Next, we studied the ability of flower infusions from both plants to inhibit aldose reductase in vitro, the protective role in the cell model of lipotoxicity, and the cytotoxic action on fibroblast cell line NIH-3T3 by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide assay. Both species are rich in phenolics; C. kousa flowers contain slightly higher amounts of phenolic acids (20.8%) and flavonoids (56.1%) than C. mas (20.2%) and (47.4%), respectively. Both extracts showed effective inhibition, expressed as half-maximal inhibitory concentration (IC50) (the concentration of inhibitor required to exhibit 50% inhibition of the enzyme reaction), of aldose reductase in non-toxic low concentrations (IC50 = 3.06 μg/mL for C. mas and IC50 = 2.49 μg/mL for C. kousa, respectively). In contrast, these concentrations of both extracts had almost no effects in the lipotoxicity cell model. To our knowledge, this study is the first report on C. mas and C. kousa flowers’ aldose reductase inhibitory activity and influence upon lipotoxicity.

Genetic parameters for milk fatty acid composition of Holstein in Korea.

ObjectiveMilk fatty acid (FA) is a main nutritional component that markedly effects human health. Intentional modification of the FA profile has the potential to improve milk quality. This study aimed at the factors affecting elevated FA levels and the estimation of the genetic parameters for milk FAs in the Korean Holstein population.MethodsTotal 885,249 repeated test-day milk records including, milk yield, saturated fatty acids (SFA), polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), total unsaturated fatty acids (TUFA), fat and protein percentages were analyzed using CombiFoss FT+ system (Foss Analytical A/S, Denmark). Genetic parameters were estimated by the restricted maximum likelihood procedure based on the repeatability model using the Wombat program.ResultsThe FA profile varies along with the lactation and the energy balance (EB). With the negative EB in early lactation, mobilization of body fat reserves elevates the desirable FA levels. As a result of that, milk quality is increased by means of nutritionally and usability aspects during the early lactation. Moreover, heritability estimates for SFA, MUFA, PUFA, TUFA were 0.33, 0.42, 0.37, 0.41 respectively. According to the parity wise heritability analysis, first parity cows had relatively lower heritability for SFAs (0.19) than later parities (0.28).ConclusionGenetic parameters indicated that FAs were under stronger genetic control. Therefore, we suggest implementing animal breeding programs towards improving the milk FA profile.

A Phenotypic Screen Identifies Calcium Overload as a Key Mechanism of β-Cell Glucolipotoxicity.

Type 2 diabetes (T2D) is caused by loss of pancreatic β-cell mass and failure of the remaining β-cells to deliver sufficient insulin to meet demand. β-Cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on β-cell function and survival, contributes to T2D-associated β-cell failure. Drugs and mechanisms that protect β-cells from GLT stress could potentially improve metabolic control in patients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected β-cells from GLT, we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. We discovered that compound A analogs decreased GLT-induced cytosolic calcium influx in islet cells, and all measured β-cell-protective effects correlated with this activity. Further studies revealed that the active compound from this series largely reversed GLT-induced global transcriptional changes. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve β-cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment.

Plasma and Tissue Bioavailability and Blood Pressure Lowering Effects of Omega-3 Polyunsaturated Fatty Acids from Commonly Eaten, Naturally Enriched, Foods

AbstractGreater consumption of oily fish, and elevated plasma levels of the long-chain marine-derived omega-3 polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have been strongly and consistently associated with a reduced incidence of heart attacks, strokes, cognitive decline, Alzheimer's disease and cancers. Despite international guidelines recommending eating oily fish at least once per week (> 250mg/day DHA and EPA), many people do not eat fish, and only a small minority of the world's population have optimal omega-3-PUFA plasma and tissue levels. Hence, in this project, we studied the bioavailability and blood pressure lowering effects of recently developed alternatives to oily fish, namely chicken-meat and eggs naturally enriched with algae-sourced omega-3-PUFAs.This was a double-blinded, controlled, randomised trial. Using a 2X2 factorial design, 161 healthy participants were randomized to eat at least 3 portions/week of omega-3-PUFA enriched (or control) chicken-meat, and to eat at least 3 omega-3-PUFA enriched (or control) eggs/week, for 6 months.Compared to control foods, eating enriched eggs, enriched chicken-meat and the combination for 6 months, resulted in plasma EPA increments (μg/g) of 3.1[2.8], 8.8[2.9]* & 4.7[2.9], plasma DHA increments (μg/g) of 5.1[3.7], 9.3[3.8]* & 13.1[3.9]*, and omega-3-index (sum of EPA + DHA as a percentage of total fatty acids in erythrocytes, %) increments of 0.9[0.3]*, 0.7[0.4]* & 1.5[0.4]*, respectively. Mean 24-hour ambulatory blood pressure (systolic/diastolic, mmHg) declined with the enriched foods 0.3[1.3]/-0.2[1.0], -0.7[1.4]/-1.4[1.0], & -1.9[1.4]/-2.9[1.0]*, respectively. Data provided as mean difference[SEM], and * denotes p < 0.05.Regular consumption of omega-3-PUFA enriched chicken-meat and eggs resulted in statistically significant and clinically relevant increases in plasma and red cell levels of DHA and EPA. Both plasma and red cell bioavailability were similar to those previously described for oily fish. Furthermore, blood pressure, an important biomarker of cardiovascular health, decreased in those eating the omega-3-PUFA enriched foods. Omega-3-PUFA enriched chicken-meat and eggs offers consumers an attractive additional alternative to eating oily fish. Unlike many lifestyle interventions, long-term large population health benefits are not dependent on willingness of people to make long-lasting dietary changes, but on the widespread availability of a range of inexpensive, commonly eaten, naturally enriched foods.

A strategy for stimulating astaxanthin and lipid production in Haematococcus pluvialis by exogenous glycerol application under low light

In large-scale outdoor cultivation, Haematococcus pluvialis cultures are frequently exposed to low light caused by continuous rain conditions. Low light significantly restricts biomass and astaxanthin accumulation, thereby decreasing astaxanthin productivity. In this study, the effects of exogenous glycerol on the growth, photosynthesis, and astaxanthin and lipid accumulation in H. pluvialis under low light were investigated. A strategy was developed for promoting astaxanthin and lipid production in H. pluvialis under low light by the application of exogenous glycerol. Under low light, exogenous glycerol did not change the biomass and photosynthetic capacity of H. pluvialis. The addition of 1 mL L−1 glycerol increased total lipid content by 36.8% by increasing the formation of fatty acids during an 11-day growth period. Furthermore, exogenous glycerol could directly accelerate astaxanthin production under low light by increasing the formation of substrates (e.g. pyruvate). Meanwhile, the elevated fatty acid level due to the addition of glycerol facilitated the esterification of astaxanthin, thereby accelerating the biosynthesis of astaxanthin indirectly, which is indicated by the fact that exogenous glycerol enhanced astaxanthin esters content significantly under low light. All these results suggest that H. pluvialis has the ability to utilize glycerol for astaxanthin and lipid production under low light.

N-acetyl cysteine attenuates oxidative stress and glutathione-dependent redox imbalance caused by high glucose/high palmitic acid treatment in pancreatic Rin-5F cells.

Elevated levels of glucose and fatty acids are the main characteristics of diabetes, obesity and other metabolic disorders, associated with increased oxidative stress, mitochondrial dysfunction and inflammation. Once the primary pathogenesis of diabetes is established, which is potentially linked to both genetic and environmental factors, hyperglycemia and hyperlipidemia exert further destructive and/or toxic effects on β-cells. The concept of glucolipotoxicity has arisen from the combination of deleterious effects of chronic elevation of glucose and fatty acid levels on pancreatic β- cell function and/or survival. Though numerous studies have been conducted in this field, the exact molecular mechanisms and causative factors still need to be established. The aim of the present work was to elucidate the molecular mechanisms of oxidative stress, and inflammatory/antioxidant responses in the presence of high concentrations of glucose/fatty acids in a cell-culture system using an insulin-secreting pancreatic β-cell line (Rin-5F) and to study the effects of the antioxidant, N-acetyl cysteine (NAC) on β-cell toxicity. In our study, we investigated the molecular mechanism of cytotoxicity in the presence of high glucose (up to 25 mM) and high palmitic acid (up to 0.3 mM) on Rin-5F cells. Our results suggest that the cellular and molecular mechanisms underlying β-cell toxicity are mediated by increased oxidative stress, imbalance of redox homeostasis, glutathione (GSH) metabolism and alterations in inflammatory responses. Pre-treatment with NAC attenuated oxidative stress and alterations in GSH metabolism associated with β-cells cytotoxicity.

Palmitate and oleate modify membrane fluidity and kinase activities of INS-1E β-cells alongside altered metabolism-secretion coupling

Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1–0.5 μM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.

196 Steroid concentrations in bovine follicular fluid are affected by metabolic stress

The negative energy balance (NEB) in dairy cows, early postpartum, is related to reduced fertility. A metabolic characteristic of NEB is elevated levels of free fatty acids (NEFA) in blood and follicular fluid with an effect on the cumulus-oocyte complex (COC; Leroy et al. 2005 Reproduction 130, 485-495, https://doi.org/10.1530/rep.1.00735; Aardema et al. 2011 Biol. Reprod. 85, 62-69, https://doi.org/10.1095/biolreprod.110.088815; Aardema et al. 2013 Biol. Reprod. 88, 164, https://doi.org/10.1095/biolreprod.112.106062). In this study, we investigated the effect of elevated NEFA levels on granulosa cells and consequently steroid production during NEB. Both 17β-oestradiol (E2) and progesterone (P4) levels in follicular fluid and oocyte competence were measured during NEB, as oocyte competence and steroid production are related (Aardema et al. 2013 Theriogenology 80, 576-583, https://doi.org/10.1016/j.theriogenology.2013.05.025). Heifers were directed towards a control (CTR; n=6) or NEB group (NEB; n=6; fasting heifer model; Jorritsma et al. 2003 Theriogenology). Cycle synchronization and superstimulation were performed as previously reported; invivo matured oocytes were, by ovariectomy at 22h after the induced LH peak, individually collected from follicles (≥8 to ≤20mm) and invitro fertilized and cultured until Day 8. Levels of nonesterified fatty acids (NEFAs) in follicular fluid were, in CTR, 80μM palmitic (C16:0), 70μM stearic (C18:0), and 100μM oleic acid (C18:1) and, in NEB, 150μM C16:0, 100μM C18:0, and 200μM C18:1 (Aardema et al. 2013 Biol. Reprod. Vol, page range). Follicular fluid of individual follicles was frozen and stored at −20°C until E2 and P4 radioimmunoassay analysis. At Day 5 and 8 of culture, embryos were scored for developmental competence, according to Aardema et al. (2013 Biol. Reprod. Vol, page range). Oocytes developing to a blastocyst all originated from follicles with, formerly in control animals defined, optimal follicular fluid levels of E2 ≤0.25 μM and P4 ≥0.26 μM (Aardema et al. 2013 Theriogenology 80, 576-583, https://doi.org/10.1016/j.theriogenology.2013.05.025). Overall E2 levels in follicular fluid in NEB appeared to be lower than those in CTR. There was a reduced positive value of 0.33 (vs. 0.49 in CTR) for predicting the competence of oocytes to develop into a blastocyst, and reduced specificity of 0.26 (vs. 0.75 in CTR) for oocytes originating from follicles with optimal steroid levels in NEB. The sensitivity and negative predictive value were 1.0 in both NEB and CTR. Levels of P4 in follicular fluid were unaffected by NEB. The lower level of E2 indicates an effect of NEB on steroid synthesis in granulosa cells. Recently, it was demonstrated that invitro exposure to monounsaturated C18:1, in contrast to saturated C16:0 or C18:0, resulted in reduced E2 production and expression of CYP19A1 in granulosa cells (Sharma 2018 Endocrine Connections 8, 493-505, https://doi.org/10.1530/EC-19-0011). These data suggest that elevated levels of NEFAs, C18:1 in particular, affect the function of granulosa cells and the follicular steroid environment of the COC, in contrast to the effect of C18:1 on the COC, which appears to protect the oocyte against lipotoxic events by saturated NEFAs by massive lipid accumulation in cumulus cells (Aardema et al. 2011 Biol. Reprod. 85, 62-69, https://doi.org/10.1095/biolreprod.110.088815; Aardema et al. 2013 Biol. Reprod. 88, 164, https://doi.org/10.1095/biolreprod.112.106062). Elevated levels of C18:1 may also result in increased lipid storage in granulosa cells and could affect the steroid synthesis of cholesterol that is also stored in lipid droplets. How elevated NEFA levels during the NEB affect granulosa cells is under investigation.

Investigation of the putative rate-limiting role of electron transfer in fatty acid desaturation using transfected HEK293T cells.

Elevated fatty acid (FA) levels contribute to severe metabolic diseases. Unbalanced oversupply of saturated FAs is particularly damaging, which renders stearoyl-CoA desaturase (SCD1) activity an important factor of resistance. A SCD1-related oxidoreductase protects cells against palmitate toxicity, so we aimed to test whether desaturase activity is limited by SCD1 itself or by the associated electron supply. Unsaturated/saturated FA ratio was markedly elevated by SCD1 overexpression while it remained unaffected by the overexpression of SCD1-related electron transfer proteins in HEK293T cells. Electron supply was not rate-limiting either in palmitate-treated cells or in cells with enhanced SCD1 expression. Our findings indicate the rate-limiting role of SCD1 itself, and that FA desaturation cannot be facilitated by reinforcing the electron supply of the enzyme.

The Cooperative Induction of CCL4 in Human Monocytic Cells by TNF-α and Palmitate Requires MyD88 and Involves MAPK/NF-κB Signaling Pathways.

Chronic low-grade inflammation, also known as metabolic inflammation, is a hallmark of obesity and parallels with the presence of elevated circulatory levels of free fatty acids and inflammatory cytokines/chemokines. CCL4/MIP-1β chemokine plays a key role in the adipose tissue monocyte recruitment. Increased circulatory levels of TNF-α, palmitate and CCL4 are co-expressed in obesity. We asked if the TNF-α/palmitate could interact cooperatively to augment the CCL4 production in human monocytic cells and macrophages. THP-1 cells/primary macrophages were co-treated with TNF-α/palmitate and CCL4 mRNA/protein expression was assessed using qRT-PCR/ELISA. TLR4 siRNA, a TLR4 receptor-blocking antibody, XBlue™-defMyD cells and pathway inhibitors were used to decipher the signaling mechanisms. We found that TNF-α/palmitate co-stimulation augmented the CCL4 expression in monocytic cells and macrophages compared to controls (p < 0.05). TLR4 suppression or neutralization abrogated the CCL4 expression in monocytic cells. Notably, CCL4 cooperative induction in monocytic cells was: (1) Markedly less in MyD88-deficient cells, (2) IRF3 independent, (3) clathrin dependent and (4) associated with the signaling mechanism involving ERK1/2, c-Jun, JNK and NF-κB. In conclusion, TNF-α/palmitate co-stimulation promotes the CCL4 expression in human monocytic cells through the mechanism involving a TLR4-MyD88 axis and MAPK/NF-κB pathways. These findings unravel a novel mechanism of the cooperative induction of CCL4 by TNF-α and palmitate which could be relevant to metabolic inflammation.

Alcohol-induced lipid dysregulation impairs glycolytic responses to LPS in alveolar macrophages.

Several conditions are marked by increased susceptibility to, and enhanced severity of, bacterial infections. Alcohol use disorder, one of these conditions, is known to predispose to bacterial pneumonia by suppressing the lung's innate immune system, and more specifically by disrupting critical alveolar macrophage (AM) functions. Recently, we established that chronic ethanol consumption also perturbs surfactant lipid homeostasis in the lung and that elevated concentrations of free fatty acids contribute to blocking essential AM functions, such as agonist-induced cytokine expression. In this study, we extend these observations by showing that elevated free fatty acid levels impair metabolic responses to lipopolysaccharide (LPS) in AMs. In particular, we show that the glycolytic reprogramming characteristic of LPS-stimulated AMs is blunted by the saturated fatty acid palmitate, whereas oleate, an unsaturated fatty acid, or ethanol alone, had no effect on this adaptive metabolic response. Additionally, we found that elevated concentrations of palmitate induced mitochondrial oxidative stress and that glycolytic reprogramming and cytokine production to LPS could be partially restored in AMs by either pharmacologically blocking palmitate entry into mitochondria or administering a mitochondrial-specific antioxidant. Taken together, these findings suggest that alcohol and elevated levels of saturated fatty acids conspire to impair pulmonary innate immunity by altering metabolic responses in AMs. Additionally, our findings suggest that targeting the mechanisms involved in fatty acid metabolism can restore pulmonary immunity and possibly limit bacterial pneumonia in individuals with alcohol use disorder.

Accelerated phosphatidylcholine turnover in macrophages promotes adipose tissue inflammation in obesity.

White adipose tissue (WAT) inflammation contributes to the development of insulin resistance in obesity. While the role of adipose tissue macrophage (ATM) pro-inflammatory signalling in the development of insulin resistance has been established, it is less clear how WAT inflammation is initiated. Here, we show that ATMs isolated from obese mice and humans exhibit markers of increased rate of de novo phosphatidylcholine (PC) biosynthesis. Macrophage-specific knockout of phosphocholine cytidylyltransferase A (CCTα), the rate-limiting enzyme of de novo PC biosynthesis pathway, alleviated obesity-induced WAT inflammation and insulin resistance. Mechanistically, CCTα-deficient macrophages showed reduced ER stress and inflammation in response to palmitate. Surprisingly, this was not due to lower exogenous palmitate incorporation into cellular PCs. Instead, CCTα-null macrophages had lower membrane PC turnover, leading to elevated membrane polyunsaturated fatty acid levels that negated the pro-inflammatory effects of palmitate. Our results reveal a causal link between obesity-associated increase in de novo PC synthesis, accelerated PC turnover and pro-inflammatory activation of ATMs.

2500-PUB: Mir-302 Protects against Glucolipotoxicity-Induced ß-Cell Dysfunction and Apoptosis through the Regulation of MST1 and PDX1

Evidence is now revealed that the cytotoxic effect caused by elevated levels of plasma glucose and free fatty acids, also called as glucolipotoxicity, is particularly harmful for pancreatic β-cell. In fact, the mammalian Ste20-like kinase 1 (MST1) can mediate glucolipotoxicity-induced β-cell damages by degradation of pancreatic and duodenal homeobox 1 (PDX-1), which is known to play an essential role in maintaining β-cell function and survival. Interestingly, the activation of the pluripotency-related microRNA cluster miR-302 can protect β-cells from glucolipotoxicity-induced apoptosis. However, few studies have examined the related mechanism in detail. In the present study, we investigated the protective roles of miR-302 on rat pancreatic RINm5F β-cells, and demonstrated that MST1 was confirmed as a target of miR-302. As a result, the activation of miR-302restored the expression of PDX1 by inactivating MST1 thus ameliorating glucolipotoxicity-induced β-cell impairments. In addition, miR-302 also upregulated endogenous antioxidant signaling and autophagic activities during glucolipotoxicity, suggesting the anti-aging process may be also involved in miR-302-mediated protection. In conclusion, our study suggests that miR-302 may serve as a potential target for developing new diagnoses and therapies to reduce glucolipotoxicity in β-cells. Disclosure C. Huang: None. H. Li: None. E. Kornelius: None. Y. Yang: None. H. Ho: None. Y. Bai: None. C. Kuo: None. C. Peng: None. C. Lin: None. Funding Ministry of Science and Technology of Taiwan (105-2314-B-040-013-MY3)

Effect of liraglutide on estimates of lipolysis and lipid oxidation in obese patients with stable coronary artery disease and newly diagnosed type 2 diabetes: A randomized trial.

Elevated levels of non-esterified fatty acids (NEFA) play a role in insulin resistance, impaired beta-cell function and they are a denominator of the abnormal atherogenic lipid profile that characterizes obese patients with type 2 diabetes (T2DM). We hypothesized that the GLP-1 receptor agonist liraglutide, in combination with metformin, would reduce lipolysis. In a randomized, double-blind, placebo-controlled, cross-over trial, 41 T2DM patients with coronary artery disease were randomized and treated with liraglutide-metformin vs placebo-metformin during 12- + 12-week periods with a wash-out period of at least 2 weeks before and between the intervention periods. NEFA kinetics were estimated using the Boston Minimal Model of NEFA metabolism, with plasma NEFA and glucose levels measured during a standard 180-minute frequently sampled intravenous glucose tolerance test. Liraglutide-metformin reduced estimates of lipolysis. Furthermore, placebo-metformin increased estimates of lipid oxidation, while treatment with liraglutide eliminated this effect. We conclude that liraglutide exerts a clinically relevant reduction in estimates of lipolysis and lipid oxidation which is explained, in part, by improved insulin secretion, as revealed by an intravenous glucose tolerance test.