Saturated Fatty Acid Palmitate Research Articles

Deconstructing metabolic inflammation using cellular systems.

Over the past years, we have embarked in a systematic analysis of the effect of obesity or fatty acids on circulating monocytes, microvascular endothelial cells, macrophages, and skeletal muscle cells. With the use of cell culture strategies, we have deconstructed complex physiological systems and then reconstructed "partial equations" to better understand cell-to-cell communication. Through these approaches, we identified that in high saturated fat environments, cell-autonomous proinflammatory pathways are activated in monocytes and endothelial cells, promoting monocyte adhesion and transmigration. We think of this as a paradigm of the conditions promoting immune cell infiltration into tissues during obesity. In concert, it is possible that muscle and adipose tissue secrete immune cell chemoattractants, and indeed, our tissue culture reconstructions reveal that myotubes treated with the saturated fatty acid palmitate, but not the unsaturated fatty acid palmitoleate, release nucleotides that attract monocytes and other compounds that promote proinflammatory classically activated "(M1)-like" polarization in macrophages. In addition, palmitate directly triggers an M1-like macrophage phenotype, and secretions from these activated macrophages confer insulin resistance to target muscle cells. Together, these studies suggest that in pathophysiological conditions of excess fat, the muscle, endothelial and immune cells engage in a synergistic crosstalk that exacerbates tissue inflammation, leukocyte infiltration, polarization, and consequent insulin resistance.

The physical chemical and biological features of triglycerides. The cell absorption of functionally different palmitic+oleic lipoproteins of very low and density and linoleic+linolenic lipoproteins of low density.

The earlier insulin-independent low-density lipoproteins and more late insulin-dependent very low-density lipoproteins implement different functions at the stages of phylogenesis. The disorder of biological function of trophology, alteration of fatty acids in triglycerides, prevalence of palmitic very low-density lipoproteins over oleic very low-density lipoproteins supply mitochondria of cells with non-optimal substrate - palmitic saturated fatty acid for gaining energy, ATP synthesis. Physiologically, cells implement oleic alternative of fatty acids metabolism, oxidizing mainly ω-9 endogenous oleic mono-unsaturated fatty acid. The pathology of low density lipoproteins is primary deficiency of poly-unsaturated fatty acids in cells, atherosclerosis and atheromotosis of intima of arteries of elastic type with development of dense plaques from poly-unsaturated fatty acids in the form of polyethers of cholesterol. The pathology of very low-density lipoproteins includes: a) syndrome of resistance to insulin; b) pathology of phylogenetically earlier insulin-independent visceral fatty tissue - metabolic syndrome; c) pathology of phylogenetically later insulin-dependent subcutaneous adipocytes - obesity; d) secondary atherosclerosis, under cumulation of palmitic low-density lipoproteins in blood with development of atherothrombosis of intima of arteries, soft plaques rich with triglycerides. As for the prevention of disorders of transfer of fatty acids to very low-density lipoproteins and low-density lipoproteins is common in many ways - minimization of aphysiological effect of surplus amount of food, biological function of diet. The prevention at the level of population includes: a) maximal limitation of content of palmitic saturated fatty acid in food; b) moderate increasing of polysaturated fatty acids, ω-3 poly-saturated fatty acids predominantly; c) increasing of physical activity. The pharmaceuticals are not provided by biology in primary prevention of metabolic pandemics under aphysiological impact of environment factors.

How surplus of palmitic fatty acid in food initiates hypertriglyceridemia, increases cholesterol of low density lipoproteins, triggers atherosclerosis and develops atheromatosis.

In phylogenesis, the first transfer of all fatty acids to cells is implemented by high density lipoproteins. Later, unsaturated and polyene fatty acids are transferred to cell by low density lipoproteins. The insulin-depended cells absorb palmitic saturated fatty acid, oleic mono-unsaturated fatty acid and of the same name triglycerides in very low density lipoproteins. The hepatocytes secrete palmitic, oleic and linoleic very low density lipoproteins separately. In blood, under hydrolysis of triglycerides, cells absorb ligand palmitic and oleic very low density lipoproteins by force of апоЕ/В-100 endocytosis; they are not transformed into low density lipoproteins. The palmitic saturated fatty acids in the form of polyether of cholesterol turn into linoleic very low density lipoproteins from high density lipoproteins at impact of protein transferring polyene ethers of cholesterol. They transform very low density lipoproteins into low density lipoproteins of the same name; the cells absorb them by force of апоЕ/В-100 endocytosis. In physiological sense, amount of oleic very low density lipoproteins are always more than palmitic of very low density lipoproteins. Under syndrome of insulin-resistance there is no transformation of palmitic saturated fatty acid synthesized from glucose in vivo into oleic mono-saturated fatty acid. The hepatocytes secrete into blood mainly palmitic very low density lipoproteins which amount exceeds oleic very low density lipoproteins. Under slow hydrolysis in blood, main mass of palmitic very low density lipoproteins becomes palmitic low density lipoproteins. These very lipoproteins initiate hyperlipidemia, increase content of cholesterol of cholesterol-low density lipoproteins, lower cholesterol-high density lipoproteins, decrease bio-availability of polyene fatty acids for cells, trigger development of atherosclerosis and formation of atheromatosis in intima of arteries. The aphysiologic effect of surplus of palmitic saturated fatty acid in vivo and triglycerides of the same name can't be eliminated under increasing of content of ω-3 polyene fatty acids in food and effect of statines. All this is to be rationally applied in prevention of hypertriglyceridemia, atherosclerosis, atheromatosis of coronary arteries, ischemic heart disease and myocardium infarction.

The atherosclerosis and atheromatosis as two etiologically different aphysiological processes at single pathogenesis. Atheromatosis and pathogenetic role of insulin.

It is supposed that at stages of phylogenesis seven biological functions was developed: 1) biological function of trophology; 2) homeostasis; 3) endoecology; 4) adaptation; 5) continuation of species; 6) locomotion; 7) cognitive function, including intellect. The function of trophology (feeding) is implemented by two biological reactions: exophilia - external feeding and endophilia - internal feeding. The function of endoecology prevents exceeding of upper limit of physiological interval by no substrate, catabolites and endogenous phlogogens. It is implemented by two biological functions: excretion and inflammation. The etiological factors of atherosclerosis are the following ones. The oleic mono-saturated fatty acid in chemical reactions is by far more active than palmitic fatty acid. In the ocean, all animals were carnivorous (piscivorous); species Homo Sapiens, in millions of years of life on dry land, forcedly became a herbivorousone. The main role in development of herbivorous animals belongs to insulin; the hormone regulating in the first-place metabolism of fatty acids, expresses transmutation of all endogenously synthesized from glucose palmitic saturated fatty acid in oleic monosaturated fatty acid. The late in phylogenesis insulin can't initiate transmutation of exogenous palmitic saturated fatty acid of food into oleic mono-saturated fatty acid. Under effect of insulin in vivo an active oleic type of metabolism of fatty acids is developed; and outside if effect of insulin palmitic type of metabolism of fatty acids is developed. In the ocean, synthesis of active eicosanoids occurs from ῳ-3 polyene fatty acids; there is no such fatty acids in dry land. The basis of pathogenesis of atherosclerosis is feeding of large amount of carnivorous (meat) food by phylogenetically herbivorous Homo Sapiens. This way a deficiency of palmitic saturated fatty acids in cells if developed blocking their bio-accessibility. The lipoproteins of low density are not developed in the initiated by insulin transfer of oleic triglycerides to cells in oleic apoE/B-100 lipoproteins of very low density and their absorption by cells. The transfer of triglycerides into lipoproteins of very low density is blocked under slow processes of transmutation of palmitic lipoproteins of very low density into lipoproteins of low density, retention cumulation of lipoproteins of low density in blood. Only because of partial utilization by monocytes of non-ligand palmitic lipoproteins of very low density →lipoproteins of low density that occurs in the intima of arteries of elastic type, atheromotosis is developed. The atheromotosis masses of intima are first of all interim catabolites of polyene fatty acids; the cells could not to absorb them by apoB-100-endocytosis in the content of lipoproteins of low density. The atherosclerosis, hyperlipoproteinemia, high content of lipoproteins of low density in blood and deficiency of polyene fatty acids in cells are a result of disorder of trophology function; the atheromotosis of arteries is only partial implementation of endoecology function.

The integrated etiology and separate pathogenesis of atherosclerosis and atheromatosis. The differences of fatty acids transfer in lipoproteins of herbivorous and carnivorous animals.

According phylogenetic theory of general pathology, overconsumption of meat food by herbivorous animals always results in atherosclerosis and atheromatosis of intima of arteries. The etiological factors of atherosclerosis, atheromatosis in vivo: a) absorption by cells of polyene fatty acids in anoB-100 lipoproteins of low density; b) impossibility of converting exogenous palmitic saturated fatty acids into mono-unsaturated oleic fatty acid; c) monocytes-macrophages in intima inactively hydrolyze polyene fatty acids esterified by alcohol cholesterol. The disorder of biological function of trophology (nutrition), biological reaction of exotrophy (external nutrition) and aphysiologically high content of palmitic unsaturated fatty acids and alcohol cholesterol in food become a pathogenic factors. The key stage of pathogenesis is formation in blood of non-ligand palmitic lipoproteins of very low density. At that: a) how to utilize in vivo non-ligand palmitic lipoproteins of very low density; it is possible under activation of biological function of endoecology, biological reaction of inflammation, formation of atheromatosis of intima of arteries and b) how to prolong function to cells at impossibility of of absorbing from inter-cellular medium polyene fatty acids; this is a foundation of atherosclerosis, disorders of biological function of adaptation, biological reaction of compensation. The primary prevention of myocardium infarction requires elimination of consumption of surplus amount of animal food. At low content of palmitic saturated fatty acids in food insulin forms optimal oleic alternative of metabolism of fatty acids supporting high "kinetic parameters" of organism and effective synthesis of ATP. According single pathogenesis of atherosclerosis and atheromatosis it is necessary to prevent development of non-ligand palmitic lipoproteins of very low density in blood. Without them no development of both atherosclerosis and atheromatosis is possible.

The nicotinic acid as an inhibitor of lipolysis in the phylogenetically early visceral cells; insulin also blocks hydrolysis of triglycerides in the phylogenetically late adipocytes.

It is valid to consider effect of nicotinic acid as an insulin-mimetic one. The uniformity of biologic effect of exogenous nicotinic acid and endogenous insulin permits to become aware that a) the hypo-lipidemic activity of insulin, inhibition of lipolysis in phylogenetically late insulin-dependent adipocytes and decreasing of content of unesterified fatty acids in blood plasma are considered as a basis of hypoglycemic effect of insulin; b) nicotinic acid similar to insulin blocks lipolysis too but in hormoneindependent visceral fatty cells. The counter-insular effect is manifested in vivo by exogenous and endogenous palmitic saturated fatty acid by force of physical chemical characteristics. The biological role of insulin consists in regulation of metabolism of fatty acids mainly unesterified fatty acids and in absorption of glucose by all insulin-dependent cells. It is supposed that contractile cells (myocytes and cardiomyocytes) cumulate glycogen for implementing glucose as a substrate in synthesis in situ de novo of ῲ-9 oleic mono-saturated fatty acid. The insulin initiates synthesis because mitochondria process this mono-saturated fatty to β-oxidation with the highest constant of reaction velocity. This is conditioned by physical chemical characteristics of oleic mono-saturated fatty acid, positioning of double bond in the chain of fatty acid; this ensures maximal efficiency of ATP gaining. In phylogenesis, the low efficient palmitic alternative of metabolism of fatty acids is formed the earliest. At the later stages of phylogenesis the cells, under becoming of motion function, worked out more efficient alternative of ATP synthesis from exogenous ῲ-9 oleic monosaturated fatty acid. At the late stages of phylogenesis, under becoming of biological function of locomotion (motion at the expense of contraction of cross-striated skeletal myocytes) insulin began to activate absorption of glucose by cells with subsequent synthesis of endogenous ῲ-9 oleic mono-saturated fatty acid out of it. The aphysiological effect of environment factors in the form of derangement of biological function of trophology (feeding), surplus of palmitic unesterified fatty acids in food serve as a main cause of such a high rate of functional derangement - insulin resistance syndrome in populations. Hence, there is no reason to call it diabetes mellitus type II.

Glucolipotoxicity diminishes cardiomyocyte TFEB and inhibits lysosomal autophagy during obesity and diabetes

Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.

MiR-1271 upregulated by saturated fatty acid palmitate provokes impaired insulin signaling by repressing INSR and IRS-1 expression in HepG2 cells

Dietary saturated fatty acids (SFA) in excess not only induce hepatic insulin resistance, but also result in type 2 diabetes (T2DM). Although microRNAs (miRNAs) participate widely in the pathogenesis of a range of diseases through the suppression of target gene expression at the post-transcriptional level, the implications of SFA-induced miRNAs in the dysregulation of metabolism, particularly in the development of insulin resistance, are largely unclear. SFA palmitate provoked an impairment of insulin signaling in HepG2 cells via a reduction in the expression of INSR and IRS-1 protein. The significant upregulation of miR-1271, which was presumed to target INSR and IRS-1 3′UTRs, was observed in the palmitate-treated HepG2 cells. Using a reporter gene assay, miR-1271 authentically targeted the 3′UTRs of INSR and IRS-1. Furthermore, the overexpression of miR-1271 caused a substantial decrease in INSR and IRS-1 expression, which led to an impairment in insulin signaling and glycogen metabolism. Therefore, these findings suggest that the induction of miR-1271 by SFA palmitate promotes the development of insulin resistance by targeting INSR and IRS-1 in hepatocytes.

Fatty acids stimulate insulin secretion from human pancreatic islets at fasting glucose concentrations via mitochondria-dependent and -independent mechanisms.

BackgroundFree fatty acids (FFAs) acutely stimulate insulin secretion from pancreatic islets. Conflicting results have been presented regarding this effect at non-stimulatory glucose concentration, however. The aim of our study was to investigate how long-chain FFAs affect insulin secretion from isolated human pancreatic islets in the presence of physiologically fasting glucose concentrations and to explore the contribution of mitochondria to the effects on secretion.MethodsInsulin secretion from human pancreatic islets was measured from short-term static incubation or perfusion system at fasting glucose concentration (5.5 mM) with or without 4 different FFAs (palmitate, palmitoleate, stearate, and oleate). The contribution of mitochondrial metabolism to the effects of fatty acid-stimulated insulin secretion was explored.ResultsThe average increase in insulin secretion, measured from statically incubated and dynamically perifused human islets, was about 2-fold for saturated free fatty acids (SFAs) (palmitate and stearate) and 3-fold for mono-unsaturated free fatty acids (MUFAs) (palmitoleate and oleate) compared with 5.5 mmol/l glucose alone. Accordingly, MUFAs induced 50 % and SFAs 20 % higher levels of oxygen consumption compared with islets exposed to 5.5 mmol/l glucose alone. The effect was due to increased glycolysis. When glucose was omitted from the medium, addition of the FFAs did not affect oxygen consumption. However, the FFAs still stimulated insulin secretion from the islets although secretion was more than halved. The mitochondria-independent action was via fatty acid metabolism and FFAR1/GPR40 signaling.ConclusionsThe findings suggest that long-chain FFAs acutely induce insulin secretion from human islets at physiologically fasting glucose concentrations, with MUFAs being more potent than SFAs, and that this effect is associated with increased glycolytic flux and mitochondrial respiration.

AMPK-independent inhibition of human macrophage ER stress response by AICAR.

Obesity-associated insulin resistance is driven by inflammatory processes in response to metabolic overload. Obesity-associated inflammation can be recapitulated in cell culture by exposing macrophages to saturated fatty acids (SFA), and endoplasmic reticulum (ER) stress responses essentially contribute to pro-inflammatory signalling. AMP-activated protein kinase (AMPK) is a central metabolic regulator with established anti-inflammatory actions. Whether pharmacological AMPK activation suppresses SFA-induced inflammation in a human system is unclear. In a setting of hypoxia-potentiated inflammation induced by SFA palmitate, we found that the AMP-mimetic AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) potently suppressed upregulation of ER stress marker mRNAs and pro-inflammatory cytokines. Furthermore, AICAR inhibited macrophage ER stress responses triggered by ER-stressors thapsigargin or tunicamycin. Surprisingly, AICAR acted independent of AMPK or AICAR conversion to 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl monophosphate (ZMP) while requiring intracellular uptake via the equilibrative nucleoside transporter (ENT) ENT1 or the concentrative nucleoside transporter (CNT) CNT3. AICAR did not affect the initiation of the ER stress response, but inhibited the expression of major ER stress transcriptional effectors. Furthermore, AICAR inhibited autophosphorylation of the ER stress sensor inositol-requiring enzyme 1α (IRE1α), while activating its endoribonuclease activity in vitro. Our results suggest that AMPK-independent inhibition of ER stress responses contributes to anti-inflammatory and anti-diabetic effects of AICAR.

Effect of fatty acid interaction on myoglobin oxygen affinity and triglyceride metabolism.

Recent studies have suggested myoglobin (Mb) may have other cellular functions in addition to storing and transporting O2. Indeed, NMR experiments have shown that the saturated fatty acid (FA) palmitate (PA) can interact with myoglobin (Mb) in its ligated state (MbCO and MbCN) but does not interact with Mb in its deoxygenated state. The observation has led to the hypothesis that Mb can also serve as a fatty acid transporter. The present study further investigates fatty acid interaction with the physiological states of Mb using the more soluble but unsaturated fatty acid, oleic acid (OA). OA binds to MbCO but does not bind to deoxy Mb. OA binding to Mb, however, does not alter its O2 affinity. Without any Mb, muscle has a significantly lower level of triglyceride (TG). In Mb knock-out (MbKO) mice, both heart and skeletal muscles have lower level of TG relative to the control mice. Training further decreases the relative TG in the MbKO skeletal muscle. Nevertheless, the absence of Mb and lower TG level in muscle does not impair the MbKO mouse performance as evidenced by voluntary wheel running measurements. The results support the hypothesis of a complex physiological role for Mb, especially with respect to fatty acid metabolism.

Endothelial FoxO proteins impair insulin sensitivity and restrain muscle angiogenesis in response to a high-fat diet.

Skeletal muscle microvascular dysfunction contributes to disease severity in type 2 diabetes. Recent studies indicate a role for Forkhead box O (FoxO) transcription factors in modulating endothelial cell phenotype. We hypothesized that a high-fat (HF) diet generates a dysfunctional vascular niche through an increased expression of endothelial FoxO. FoxO1 protein increased (+130%) in the skeletal muscle capillaries from HF compared to normal chow-fed mice. FoxO1 protein was significantly elevated in cultured endothelial cells exposed to the saturated fatty acid palmitate or the proinflammatory cytokine TNF-α. In HF-fed mice, endothelium-directed depletion of FoxO1/3/4 (FoxO(Δ)) improved insulin sensitivity (+110%) compared to that of the controls (FoxO(L/L)). The number of skeletal muscle capillaries increased significantly in the HF-FoxO(Δ) mice. Transcript profiling of skeletal muscle identified significant increases in genes associated with angiogenesis and lipid metabolism in HF-FoxO(Δ) vs. HF-FoxO(L/L) mice. HF-FoxO(Δ) muscle also was characterized by a decrease in inflammation-related genes and an enriched M2 macrophage signature. We conclude that endothelial FoxO proteins promote insulin resistance in HF diet, which may in part result from FoxO proteins establishing an antiangiogenic and proinflammatory microenvironment within skeletal muscle. These findings provide mechanistic insight into the development of microvascular dysfunction in the progression of type 2 diabetes.-Nwadozi, E., Roudier, E., Rullman, E., Tharmalingam, S., Liu, H.-Y., Gustafsson, T., Haas, T. L. Endothelial FoxO proteins impair insulin sensitivity and restrain muscle angiogenesis in response to a high-fat diet.

Palmitate-induced changes in energy demand cause reallocation of ATP supply in rat and human skeletal muscle cells

Mitochondrial dysfunction has been associated with obesity-related muscle insulin resistance, but the causality of this association is controversial. The notion that mitochondrial oxidative capacity may be insufficient to deal appropriately with excessive nutrient loads is for example disputed. Effective mitochondrial capacity is indirectly, but largely determined by ATP-consuming processes because skeletal muscle energy metabolism is mostly controlled by ATP demand. Probing the bioenergetics of rat and human myoblasts in real time we show here that the saturated fatty acid palmitate lowers the rate and coupling efficiency of oxidative phosphorylation under conditions it causes insulin resistance. Stearate affects the bioenergetic parameters similarly, whereas oleate and linoleate tend to decrease the rate but not the efficiency of ATP synthesis. Importantly, we reveal that palmitate influences how oxidative ATP supply is used to fuel ATP-consuming processes. Direct measurement of newly made protein demonstrates that palmitate lowers the rate of de novo protein synthesis by more than 30%. The anticipated decrease of energy demand linked to protein synthesis is confirmed by attenuated cycloheximide-sensitivity of mitochondrial respiratory activity used to make ATP. This indirect measure of ATP turnover indicates that palmitate lowers ATP supply reserved for protein synthesis by at least 40%. This decrease is also provoked by stearate, oleate and linoleate, albeit to a lesser extent. Moreover, palmitate lowers ATP supply for sodium pump activity by 60–70% and, in human cells, decreases ATP supply for DNA/RNA synthesis by almost three-quarters. These novel fatty acid effects on energy expenditure inform the ‘mitochondrial insufficiency’ debate.

Glutamine Modulates Macrophage Lipotoxicity.

Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli.

Track1: From genes to cells

The saturated fatty acid palmitate (PAL) is now recognized as an inducer of inflammation in many types of cells, including adipocytes and muscle cells, whereas n-3 polyunsaturated fatty acids (n-3PUFA) have anti-inflammatory properties. Meanwhile, PAL induces muscle insulin resistance (IR) and n-3PUFA are suggested to reverse it. We investigated n-3PUFA effects on palmitate-induced inflammation and the potential link between inflammation, P38MAPK activation and IR in C2C12 myotubes. After 16 hours incubation with 500µM PAL without or with SB203580, a specific inhibitor of p38MAPK, 50µM of alpha linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), myotubes were harvested and submitted to mRNA quantification or immunoblotting. IL-6, TNF-a and COX-2 mRNA levels were significantly increased by PAL treatment and reversed by EPA and DHA. Inhibition of p38MAPK significantly prevented the effect of PAL on these regulations although it had no significant effect on PAL-induced IR. Additional study should be performed to explore the involvement of NFKB signalling. Our results suggested that p38MAPK activation by PAL is crucial to induce inflammation in C212 muscle cells, independently of IR. Among n-3PUFA, only EPA and DHA reduced p38MAPK activation and improved IR. Additional studies are currently performed to explore the involvement of NFKB signalling in the effect of PAL on myotube inflammation and the impact of n-3 PUFA on this pathway

ATGL Is Not Required for Oleate Protection from Lipotoxicity in NIH3T3 Cells

The long chain, saturated fatty acid palmitate is toxic to cultured cells. However, co‐supplementation with monounsaturated oleate promotes storage of excess palmitate in lipid droplets and prevents cell death. We tested whether events downstream of lipid storage in lipid droplets contribute to oleate protection from lipotoxicity. Specifically, we asked whether protection from lipotoxicity requires adipose triglyceride lipase (ATGL) or sirtuin 1 (SIRT1) activity. We found that oleate continues to prevent palmitate induction of caspase 3/7 activity in NIH3T3 cells in the presence of ATGListatin, an inhibitor of ATGL. Moreover, oleate and palmitate co‐supplementation does not increase the activity of SIRT1, a transcriptional co‐activator downstream of ATGL, nor does it lead to ATGL‐dependent changes in expression of ACOT1, ACSL1, or CPT1α mRNA. Thus, our results fail to support a role for ATGL in oleate protection from lipotoxicity in NIH3T3 cells. Additional pathways through which oleate protects a cell from lipotoxicity should be explored.Support or Funding InformationThis research was funded by the Arnold and Mabel Beckman Foundation and St. Olaf College CURI.

Induction of Gnrh mRNA expression by the ω-3 polyunsaturated fatty acid docosahexaenoic acid and the saturated fatty acid palmitate in a GnRH-synthesizing neuronal cell model, mHypoA-GnRH/GFP

Gonadotropin-releasing hormone (GnRH) neurons coordinate reproduction. However, whether GnRH neurons directly sense free fatty acids (FFAs) is unknown. We investigated the individual effects of the FFAs docosahexaenoic acid (DHA), palmitate, palmitoleate, and oleate (100 μM each) on Gnrh mRNA expression in the mHypoA-GnRH/GFP neuronal cell model. We report that 2 h exposure to palmitate or DHA increases Gnrh transcription. Using the inhibitors AH7614, K252c, U0126, wortmannin, and LY294002, we demonstrate that the effect of DHA is mediated through GPR120 to downstream PKC/MAPK and PI3K signaling. Our results indicate that the effect of palmitate may depend on palmitoyl-coA synthesis and PI3K signaling. Finally, we demonstrate that both DHA and palmitate increase Gnrh enhancer-derived RNA levels. Overall, these studies provide evidence that GnRH neurons directly sense FFAs. This will advance our understanding of the mechanisms underlying FFA sensing in the brain and provides insight into the links between nutrition and reproductive function.

PKR is not obligatory for high-fat diet-induced obesity and its associated metabolic and inflammatory complications.

Protein kinase R (PKR) has previously been suggested to mediate many of the deleterious consequences of a high-fat diet (HFD). However, previous studies have observed substantial phenotypic variability when examining the metabolic consequences of PKR deletion. Accordingly, herein, we have re-examined the role of PKR in the development of obesity and its associated metabolic complications in vivo as well as its putative lipid-sensing role in vitro. Here we show that the deletion of PKR does not affect HFD-induced obesity, hepatic steatosis or glucose metabolism, and only modestly affects adipose tissue inflammation. Treatment with the saturated fatty acid palmitate in vitro induced comparable levels of inflammation in WT and PKR KO macrophages, demonstrating that PKR is not necessary for the sensing of pro-inflammatory lipids. These results challenge the proposed role for PKR in obesity, its associated metabolic complications and its role in lipid-induced inflammation.

Inhibition of NAPDH Oxidase 2 (NOX2) Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS) in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.

Involvement of Sphingosine 1-Phosphate in Palmitate-Induced Non-Alcoholic Fatty Liver Disease

Background/Aims: Ectopic lipid accumulation in hepatocytes has been identified as a risk factor for the progression of liver fibrosis and is strongly associated with obesity. In particular, the saturated fatty acid palmitate is involved in initiation of liver fibrosis via formation of secondary metabolites by hepatocytes that in turn activate hepatic stellate cells (HSCs) in a paracrine manner. Methods: α-smooth muscle actin-expression (α-SMA) as a marker of liver fibrosis was investigated via western blot analysis and immunofluorescence microscopy in HSCs (LX-2). Sphingolipid metabolism and the generation of the bioactive secondary metabolite sphingosine 1-phosphate (S1P) in response to palmitate were analyzed by LC-MS/MS in hepatocytes (HepG2). To identify the molecular mechanism involved in the progression of liver fibrosis real-time PCR analysis and pharmacological modulation of S1P receptors were performed. Results: Palmitate oversupply increased intra- and extracellular S1P-concentrations in hepatocytes. Conditioned medium from HepG2 cells initiated fibrosis by enhancing α-SMA-expression in LX-2 in a S1P-dependent manner. In accordance, fibrotic response in the presence of S1P was also observed in HSCs. Pharmacological inhibition of S1P receptors demonstrated that S1P₃ is the crucial receptor subtype involved in this process. Conclusion: S1P is synthesized in hepatocytes in response to palmitate and released into the extracellular environment leading to an activation of HSCs via the S1P₃ receptor.