Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes

Although fatty acids enhance preadipocyte differentiation in the presence of adequate hormone cocktails, little is known regarding their effects in the absence of these hormones. We have now shown that palmitate, a common long-chain saturated fatty acid, induced apoptosis in both mouse 3T3-L1 and rat primary preadipocytes grown in a normal serum-containing medium. Treatment of preadipocytes with palmitate induced multiple endoplasmic reticulum (ER) stress responses, evidenced by increased protein content of CHOP and GRP78 and splicing of XBP-1 mRNA, as well as altered phosphorylation of eIF2alpha and increased phosphorylation of JNK and Erk1/2. Intriguingly, palmitate induced an early activation of Akt but diminished both Akt activation and its protein mass after prolonged incubation (>6 h). In association with these changes, palmitate reduced expression of beta-catenin and its downstream target, c-Myc and cyclin D1, two key prosurvival proteins. Overexpression of constitutively active Akt did not block the apoptotic effect of palmitate. Cotreatment with unsaturated fatty acids (oleate, linoleate) or with LiCl (a glycogen synthase kinase-3beta inhibitor) attenuated the palmitate-induced apoptosis. Subsequent analysis suggested that the unsaturated fatty acids probably counteracted palmitate by reducing, not eliminating, ER stress, whereas LiCl probably improved viability by activating the Wnt signaling pathway. Cotreatment of palmitate with a standard adipogenic hormone cocktail also abolished the apoptotic effect and promoted adipocyte differentiation. Collectively, our results suggest that palmitate causes multiple cellular stresses that may lead to apoptosis in preadipocytes in the absence of adipogenic stimuli, highlighting the importance of exogenous hormones in directing cell fate in response to increased fatty acid influx.     

Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells

Accumulation of lipids in nonadipose tissues can lead to cell dysfunction and cell death, a phenomenon known as lipotoxicity. However, the signaling pathways and mechanisms linking lipid accumulation to cell death are poorly understood. The present study examined the hypothesis that saturated fatty acids disrupt endoplasmic reticulum (ER) homeostasis and promote apoptosis in liver cells via accumulation of ceramide. H4IIE liver cells were exposed to varying concentrations of saturated (palmitate or stearate) or unsaturated (oleate or linoleate) fatty acids. ER homeostasis was monitored using markers of the ER stress response pathway, including phosphorylation of IRE1alpha and eIF2alpha, splicing of XBP1 mRNA, and expression of molecular chaperone (e.g., GRP78) and proapoptotic (CCAAT/enhancer-binding protein homologous protein) genes. Apoptosis was monitored using caspase activity and DNA laddering. Palmitate and stearate induced ER stress, caspase activity, and DNA laddering. Inhibition of caspase activation prevented DNA laddering. Unsaturated fatty acids did not induce ER stress or apoptosis. Saturated fatty acids increased ceramide concentration; however, inhibition of de novo ceramide synthesis did not prevent saturated fatty acid-induced ER stress and apoptosis. Unsaturated fatty acids rescued palmitate-induced ER stress and apoptosis. These data demonstrate that saturated fatty acids disrupt ER homeostasis and induce apoptosis in liver cells via mechanisms that do not involve ceramide accumulation.     

Oleate and eicosapentaenoic acid attenuate palmitate-induced inflammation and apoptosis in renal proximal tubular cell

Free fatty acid (FFA)-bound albumin, which is filtrated through the glomeruli and reabsorbed into proximal tubular cells, is one of the crucial mediators of tubular damage in proteinuric kidney disease. In this study, we examined the role of each kind of FFA on renal tubular damage in vitro and tried to identify its molecular mechanism. In cultured proximal tubular cells, a saturated fatty acid, palmiate, increased the expression of monocyte chemoattractant protein-1 (MCP-1), but this effect was abrogated by co-incubation of monounsaturated fatty acid, oleate, or ω-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA). Palmitate led to intracellular accumulation of diacylglycerol (DAG) and subsequent activation of protein kinase C protein family. Among the several PKC inhibitors, rottlerin, a PKCθ inhibitor, prevented palmitate-induced MCP-1 expression via inactivation of NFB pathway. Overexpression of dominant-negative PKCθ also inhibited palmitate-induced activation of MCP-1 promoter. Furthermore, palmitate enhanced PKCθ-dependent mitochondrial apoptosis, which was also prevented by co-incubation with oleate or EPA through restoration of pro-survival Akt pathway. Moreover, oleate and EPA inhibited palmitate-induced PKCθ activation through the conversion of intracellular DAG to triglyceride with the restoration of diacylglycerol acyltransferase 2 expression. These results suggest that oleate and EPA have protective effects against the palmitate-induced renal tubular cell damage by inhibiting PKCθ activation.     

Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells

Here we report that in skeletal muscle cells the contribution to insulin resistance and inflammation of two common dietary long-chain fatty acids depends on the channeling of these lipids to distinct cellular metabolic fates. Exposure of cells to the saturated fatty acid palmitate led to enhanced diacylglycerol levels and the consequent activation of the protein kinase C/nuclear factor kappaB pathway, finally resulting in enhanced interleukin 6 secretion and down-regulation of the expression of genes involved in the control of the oxidative capacity of skeletal muscle (peroxisome proliferator-activated receptor (PPAR)gamma-coactivator 1alpha) and triglyceride synthesis (acyl-coenzyme A: diacylglycerol acyltransferase 2). In contrast, exposure to the monounsaturated fatty acid oleate did not lead to these changes. Interestingly, co-incubation of cells with palmitate and oleate reversed both inflammation and impairment of insulin signaling by channeling palmitate into triglycerides and by up-regulating the expression of genes involved in mitochondrial beta-oxidation, thus reducing its incorporation into diacylglycerol. Our findings support a model of cellular lipid metabolism in which oleate protects against palmitate-induced inflammation and insulin resistance in skeletal muscle cells by promoting triglyceride accumulation and mitochondrial beta-oxidation through PPARalpha- and protein kinase A-dependent mechanisms.     

Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate''s protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.     

Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6

Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of β-cell dysfunction and loss of islet mass in type 2 diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of β-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 β-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect β-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering β-cells more susceptible to saturated FA-induced ER stress and apoptosis.     

Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G

Background

In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved.

Methods

Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated.

Results

The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis.

Conclusions

Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.     

Saturated fatty acid-induced apoptosis in MDA-MB-231 breast cancer cells. A role for cardiolipin

Little is known about the biochemical basis of the action of free fatty acids (FFA) on breast cancer cell proliferation and apoptosis. Here we report that unsaturated FFAs stimulated the proliferation of human MDA-MB-231 breast cancer cells, whereas saturated FFAs inhibited it and caused apoptosis. Saturated FFA palmitate decreased the mitochondrial membrane potential and caused cytochrome c release. Palmitate-induced apoptosis was enhanced by the fat oxidation inhibitor etomoxir, whereas it was reduced by fatty-acyl CoA synthase inhibitor triacsin C. The non-metabolizable analog 2-bromopalmitate was not cytotoxic. This indicates that palmitate must be metabolized to exert its toxic effect but that its action does not involve fat oxidation. Pharmacological studies showed that the action of palmitate is not mediated via ceramides, reactive oxygen species, or changes in phosphatidylinositol 3-kinase activity. Palmitate caused early enhancement of cardiolipin turnover and decreased the levels of this mitochondrial phospholipid, which is necessary for cytochrome c retention. Cosupplementation of oleate, or increasing beta-oxidation with the AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside, both restored cardiolipin levels and blocked palmitate-induced apoptosis. Oleate was preferentially metabolized to triglycerides, and oleate cosupplementation channeled palmitate esterification processes to triglycerides. Overexpression of Bcl-2 family members blocked palmitate-induced apoptosis. The results provide evidence that a decrease in cardiolipin levels and altered mitochondrial function are involved in palmitate-induced breast cancer cell death. They also suggest that the antiapoptotic action of oleate on palmitate-induced cell death involves both restoration of cardiolipin levels and redirection of palmitate esterification processes to triglycerides.     

Ncb5or deficiency increases fatty acid catabolism and oxidative stress

The endoplasmic reticulum-associated NADH cytochrome b(5) oxidoreductase (Ncb5or) is widely distributed in animal tissues. Ncb5or(-/-) mice develop diabetes at age 7 weeks and have increased susceptibility to the diabetogenic oxidant streptozotocin. Ncb5or deficiency also results in lipoatrophy and increased hepatocyte sensitivity to cytotoxic effects of saturated fatty acids. Here we investigate the mechanisms of these phenomena in prediabetic Ncb5or(-/-) mice and find that, despite increased rates of fatty acid uptake and synthesis and higher stearoyl-CoA desaturase (SCD) expression, Ncb5or(-/-) liver accumulates less triacylglycerol (TAG) than wild type (WT). Increased fatty acid catabolism and oxidative stress are evident in Ncb5or(-/-) hepatocytes and reflect increased mitochondrial content, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) expression, fatty acid oxidation rates, oxidative stress response gene expression, and oxidized glutathione content. Ncb5or(-/-) hepatocytes readily incorporate exogenous fatty acids into TAG but accumulate more free fatty acids (FFA) and have greater palmitate-induced oxidative stress responses and cell death than WT, all of which are alleviated by co-incubation with oleate via TAG channeling. A high fat diet rich in palmitate and oleate stimulates both lipogenesis and fatty acid catabolism in Ncb5or(-/-) liver, resulting in TAG levels similar to WT but increased intracellular FFA accumulation. Hepatic SCD-specific activity is lower in Ncb5or(-/-) than in WT mice, although Ncb5or(-/-) liver has a greater increase in Scd1 mRNA and protein levels. Together, these findings suggest that increased FFA accumulation and catabolism and oxidative stress are major consequences of Ncb5or deficiency in liver.     

Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway

Accumulation of saturated fatty acids in the liver can cause nonalcoholic fatty liver disease (NAFLD). This study investigated saturated fatty acid induction of endoplasmic reticulum (ER) stress and apoptosis in human liver cells and the underlying causal mechanism. Human liver L02 and HepG2 cell lines were exposed to the saturated fatty acid sodium palmitate. MTT assay was used for cell viability, flow cytometry and Hoechst 33258 staining for apoptosis, RT-PCR for mRNA expression, and Western blot for protein expression. Silence of PRK-like ER kinase (PERK) expression in liver cells was through transient transfection of PERK shRNA. Treatment of L02 and HepG2 cells with sodium palmitate reduced cell viability through induction of apoptosis. Sodium palmitate also induced ER stress in the cells, indicated by upregulation of PERK phosphorylation and expression of BiP, ATF4, and CHOP proteins. Sodium palmitate had little effect on activating XBP-1, a common target of the other two canonical sensors of ER stress, ATF6, and IRE1. Knockdown of PERK gene expression suppressed the PERK/ATF4/CHOP signaling pathway during sodium palmitate-induced ER stress and significantly inhibited sodium palmitate-induced apoptosis in L02 and HepG2 cells. Saturated fatty acid-induced ER stress and apoptosis in these human liver cells were enacted through the PERK/ATF4/CHOP signaling pathway. Future study is warranted to investigate the role of these proteins in mediating saturated fatty acid-induced NAFLD in animal models.     

Palmitate promotes monocyte atherogenicity via de novo ceramide synthesis

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. This study investigates the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Incubation of human U937 and THP-1 monocytes with palmitate for 24h increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300μM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300μM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis.     

Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Different role of saturated and unsaturated fatty acids in beta-cell apoptosis

It is believed that free fatty acids contribute to the pathogenesis of type 2 diabetes in humans. We have recently shown that lipoapoptosis of human beta-cells is specifically induced by saturated fatty acids while unsaturated had no effect. In the present study we tested the effect of co-incubation of different saturated and unsaturated free fatty acids on lipoapoptosis in beta-cells. RIN1046-38 cells and isolated human beta-cells were incubated with combinations of saturated fatty acids (palmitate, stearate) and mono- or polyunsaturated fatty acids (palmitoleate, oleate, and linoleate). Cells were incubated for 24-72 h with 1mM fatty acids. All unsaturated fatty acids tested completely prevented palmitate- or stearate-induced apoptosis of rat and human beta-cells as assessed by flow cytometric cell cycle analysis and TUNEL assay. This might suggest that apoptosis in vivo is predominantly determined by the content of unsaturated fatty acids in a mixed fatty acid pool.     

Chronic effects of palmitate overload on nutrient-induced insulin secretion and autocrine signalling in pancreatic MIN6 beta cells

Background

Sustained exposure of pancreatic β cells to an increase in saturated fatty acids induces pleiotropic effects on β-cell function, including a reduction in stimulus-induced insulin secretion. The objective of this study was to investigate the effects of chronic over supply of palmitate upon glucose- and amino acid-stimulated insulin secretion (GSIS and AASIS, respectively) and autocrine-dependent insulin signalling with particular focus on the importance of ceramide, ERK and CaMKII signalling.

Principal Findings

GSIS and AASIS were both stimulated by >7-fold resulting in autocrine-dependent activation of protein kinase B (PKB, also known as Akt). Insulin release was dependent upon nutrient-induced activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) as their pharmacological inhibition suppressed GSIS/AASIS significantly. Chronic (48 h, 0.4 mM) palmitate treatment blunted glucose/AA-induced activation of CaMKII and ERK and caused a concomitant reduction (∼75%) in GSIS/AASIS and autocrine-dependent activation of PKB. This inhibition could not be attributed to enhanced mitochondrial fatty acid uptake/oxidation or ceramide synthesis, which were unaffected by palmitate. In contrast, diacylglycerol synthesis was elevated suggesting increased palmitate esterification rather than oxidation may contribute to impaired stimulus-secretion coupling. Consistent with this, 2-bromopalmitate, a non-oxidisable palmitate analogue, inhibited GSIS as effectively as palmitate.

Conclusions

Our results exclude changes in ceramide content or mitochondrial fatty acid handling as factors initiating palmitate-induced defects in insulin release from MIN6 β cells, but suggest that reduced CaMKII and ERK activation associated with palmitate overload may contribute to impaired stimulus-induced insulin secretion.     

CTRP5 ameliorates palmitate-induced apoptosis and insulin resistance through activation of AMPK and fatty acid oxidation

Lipotoxicity resulting from a high concentration of saturated fatty acids is closely linked to development of insulin resistance, as well as apoptosis in skeletal muscle. CTRP5, an adiponectin paralog, is known to activate AMPK and fatty acid oxidation; however, the effects of CTRP5 on palmitate-induced lipotoxicity in myocytes have not been investigated. We found that globular domain of CTRP5 (gCTRP5) prevented palmitate-induced apoptosis and insulin resistance in myocytes by inhibiting the activation of caspase-3, reactive oxygen species accumulation, and IRS-1 reduction. These beneficial effects of gCTRP5 are mainly attributed to an increase in fatty acid oxidation through phosphorylation of AMPK. These results provide a novel function of CTRP5, which may have preventive and therapeutic potential in management of obesity, insulin resistance, and type 2 diabetes mellitus.     

Unsaturated fatty acids induce non-canonical autophagy

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1^+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.     

Lovastatin does not accentuate but is rather additive to palmitate-induced apoptosis in cardiomyocytes

Fatty acids such as palmitate have been observed to induce apoptosis in cardiomyocytes but the mechanism of this cytotoxicity is unresolved. The present study sought to determine whether an aspect of fatty acid metabolism is responsible for palmitate-induced apoptosis in cardiomyocytes. As palmitate metabolism increases acetyl CoA production via increased beta oxidation within the mitochondria, we hypothesized that increased acetyl CoA entering the cholesterol biosynthesis pathway might produce intermediates or end products that would be toxic to the cell. To test this hypothesis, cardiomyocytes from embryonic chick cardiomyocytes were treated with the 3-hydroxy-3-methylgutaryl CoA (HMG-CoA) reductase inhibitor lovastatin that inhibits the cholesterol biosynthesis pathway downstream of the acetyl CoA trimerization into HMG-CoA. Lovastatin did not inhibit palmitate-induced apoptosis. Rather, lovastatin induced significant apoptosis itself and when combined with palmitate, the level of apoptosis was equal to the sum of palmitate alone and lovastatin alone. This observation suggests that palmitate and lovastatin are inducing apoptosis by two independent mechanisms. A role for mitochondrial metabolism via carnitine palmitoyl transferase (CPT) in palmitate-induced apoptosis was suggested since capric acid, a fatty acid that is metabolized within the mitochondria but does not utilize CPT-1, did not induce apoptosis. Palmitate-induced apoptosis was further related to the metabolism of saturated fatty acids as the unsaturated fatty acid oleic acid did not induce apoptosis. These data suggest that a unique feature about palmitate metabolism independent of its role in cholesterol biosynthesis is responsible for palmitate-induced apoptosis and the effects of palmitate are additive to those of lovastatin to induce cardiac apoptosis.     

Palmitate-induced apoptosis can occur through a ceramide-independent pathway

Cytotoxic accumulation of long chain fatty acids has been proposed to play an important role in the pathogenesis of diabetes mellitus and heart disease. To explore the mechanism of cellular lipotoxicity, we cultured Chinese hamster ovary cells in the presence of media supplemented with fatty acid. The saturated fatty acid palmitate, but not the monounsaturated fatty acid oleate, induced programmed cell death as determined by annexin V positivity, caspase 3 activity, and DNA laddering. De novo ceramide synthesis increased 2.4-fold with palmitate supplementation; however, this was not required for palmitate-induced apoptosis. Neither biochemical nor genetic inhibition of de novo ceramide synthesis arrested apoptosis in Chinese hamster ovary cells in response to palmitate supplementation. Rather, our data suggest that palmitate-induced apoptosis occurs through the generation of reactive oxygen species. Fluorescence of an oxidant-sensitive probe was increased 3.5-fold with palmitate supplementation indicating that production of reactive intermediates increased. In addition, palmitate-induced apoptosis was blocked by pyrrolidine dithiocarbamate and 4,5-dihydroxy-1,3-benzene-disulfonic acid, two compounds that scavenge reactive intermediates. These studies suggest that generation of reactive oxygen species, independent of ceramide synthesis, is important for the lipotoxic response and may contribute to the pathogenesis of diseases involving intracellular lipid accumulation.