Forkhead transcription factors (FoxOs) promote apoptosis of insulin-resistant macrophages during cholesterol-induced endoplasmic reticulum stress.
ABSTRACT: Endoplasmic reticulum stress increases macrophage apoptosis, contributing to the complications of atherosclerosis. Insulin-resistant macrophages are more susceptible to endoplasmic reticulum stress-associated apoptosis probably contributing to macrophage death and necrotic core formation in atherosclerotic plaques in type 2 diabetes. However, the molecular mechanisms of increased apoptosis in insulin-resistant macrophages remain unclear.The studies were performed in insulin-resistant macrophages isolated from insulin receptor knockout or ob/ob mice. Gain- or loss-of-function approaches were used to evaluate the roles of forkhead transcription factors (FoxOs) in endoplasmic reticulum stress-associated macrophage apoptosis.Insulin-resistant macrophages showed attenuated Akt activation and increased nuclear localization of FoxO1 during endoplasmic reticulum stress induced by free cholesterol loading. Overexpression of active FoxO1 or FoxO3 failed to induce apoptosis in unchallenged macrophages but exacerbated apoptosis in macrophages with an active endoplasmic reticulum stress response. Conversely, macrophages with genetic knockouts of FoxO1, -3, and -4 were resistant to apoptosis in response to endoplasmic reticulum stress. FoxO1 was shown by chromatin immunoprecipitation and promoter expression analysis to induce inhibitor of kappaBepsilon gene expression and thereby to attenuate the increase of nuclear p65 and nuclear factor-kappaB activity during endoplasmic reticulum stress, with proapoptotic and anti-inflammatory consequences.Decreased Akt and increased FoxO transcription factor activity during the endoplasmic reticulum stress response leads to increased apoptosis of insulin-resistant macrophages. FoxOs may have a dual cellular function, resulting in either proapoptotic or anti-inflammatory effects in an endoplasmic reticulum stress-modulated manner. In the complex plaque milieu, the ultimate effect is likely to be an increase in macrophage apoptosis, plaque inflammation, and destabilization.
Project description:Macrophage apoptosis is a critical process in the formation of necrotic cores in vulnerable atherosclerotic plaques. In vitro and in vivo data suggest that macrophage apoptosis in advanced atheromata may be triggered by a combination of endoplasmic reticulum stress and engagement of the type A scavenger receptor, which together induce death through a rise in cytosolic calcium and activation of toll-like receptor-4.Using both primary peritoneal macrophages and studies in advanced atheromata in vivo, we introduce signal transducer and activator of transcription-1 (STAT1) as a critical and necessary component of endoplasmic reticulum stress/type A scavenger receptor-induced macrophage apoptosis. We show that STAT1 is serine phosphorylated in macrophages subjected to type A scavenger receptor ligands and endoplasmic reticulum stress in a manner requiring cytosolic calcium, calcium/calmodulin-dependent protein kinase II, and toll-like receptor-4. Remarkably, apoptosis was inhibited by approximately 80% to 90% (P<0.05) by STAT1 deficiency or calcium/calmodulin-dependent protein kinase II inhibition. In vivo, nuclear Ser-P-STAT1 was found in macrophage-rich regions of advanced murine and human atheromata. Most important, macrophage apoptosis was decreased by 61% (P=0.034) and plaque necrosis by 34% (P=0.02) in the plaques of fat-fed low density lipoprotein receptor null Ldlr-/- mice transplanted with Stat1-/- bone marrow.STAT1 is critical for endoplasmic reticulum stress/type A scavenger receptor-induced apoptosis in primary tissue macrophages and in macrophage apoptosis in advanced atheromata. These findings suggest a potentially important role for STAT1-mediated macrophage apoptosis in atherosclerotic plaque progression.
Project description:Saturated fatty acids, such as palmitic and stearic acid, cause detrimental effects in endothelial cells and have been suggested to contribute to macrophage accumulation in adipose tissue and the vascular wall, in states of obesity and insulin resistance. Long-chain fatty acids are believed to require conversion into acyl-CoA derivatives to exert most of their detrimental effects, a reaction catalyzed by acyl-CoA synthetases (ACSLs). The objective of this study was to investigate the role of ACSL1, an ACSL isoform previously shown to mediate inflammatory effects in myeloid cells, in regulating endothelial cell responses to a saturated fatty acid-rich environment in vitro and in vivo.Saturated fatty acids caused increased inflammatory activation, endoplasmic reticulum stress, and apoptosis in mouse microvascular endothelial cells. Forced ACSL1 overexpression exacerbated the effects of saturated fatty acids on apoptosis and endoplasmic reticulum stress. However, endothelial ACSL1 deficiency did not protect against the effects of saturated fatty acids in vitro, nor did it protect insulin-resistant mice fed a saturated fatty acid-rich diet from macrophage adipose tissue accumulation or increased aortic adhesion molecule expression.Endothelial ACSL1 is not required for inflammatory and apoptotic effects of a saturated fatty acid-rich environment.
Project description:In advanced atherosclerosis, macrophage apoptosis coupled with defective phagocytic clearance of the apoptotic cells (efferocytosis) promotes plaque necrosis, which precipitates acute atherothrombotic cardiovascular events. Oxidative and endoplasmic reticulum (ER) stress in macrophages are important causes of advanced lesional macrophage apoptosis. We now show that proapoptotic oxidative/ER stress inducers trigger another stress reaction in macrophages, autophagy. Inhibition of autophagy by silencing ATG5 or other autophagy mediators enhances apoptosis and NADPH oxidase-mediated oxidative stress while at the same time rendering the apoptotic cells less well recognized by efferocytes. Most importantly, macrophage ATG5 deficiency in fat-fed Ldlr(-/-) mice increases apoptosis and oxidative stress in advanced lesional macrophages, promotes plaque necrosis, and worsens lesional efferocytosis. These findings reveal a protective process in oxidatively stressed macrophages relevant to plaque necrosis, suggesting a mechanism-based strategy to therapeutically suppress atherosclerosis progression and its clinical sequelae.
Project description:Insulin resistance renders macrophages more prone to cholesterol-induced apoptosis by promoting nuclear localization of transcription factor forkhead box transcription factor (Fox) O1. However, FoxO1 also decreases macrophage inflammation, raising the question of how the balance between proapoptotic and antiinflammatory effects is determined. We sought to identify the mechanism whereby FoxO1 dampens inflammation without promoting apoptosis. We hypothesized that nutrient-dependent FoxO1 acetylation plays a role in this process.We generated knock-in mice bearing alleles that encode constitutively deacetylated FoxO1 and studied the ex vivo response of primary peritoneal macrophages. We show that macrophages derived from mice homozygous for constitutively deacetylated FoxO1 alleles retain antiinflammatory properties in response to free cholesterol loading, without increasing apoptosis. Deacetylated FoxO1 inhibits free cholesterol-induced Akt phosphorylation and increases levels of the nuclear factor-?B precursor p105, decreasing nuclear translocation of nuclear factor-?B p65 subunit and dampening mitogen-activated protein/extracellular signal-regulated kinase activation to prevent inflammation.Deacetylated FoxO1 regulates p105 to prevent macrophage inflammation without causing apoptosis, suggesting a potential novel therapeutic approach to atherosclerosis through FoxO1 deacetylation.
Project description:The c-Jun NH2-terminal kinases (JNK) are regulated by a wide variety of cellular stresses and have been implicated in apoptotic signaling. Macrophages express 2 JNK isoforms, JNK1 and JNK2, which may have different effects on cell survival and atherosclerosis.To dissect the effect of macrophage JNK1 and JNK2 on early atherosclerosis, Ldlr(-/-) mice were reconstituted with wild-type, Jnk1(-/-), and Jnk2(-/-) hematopoietic cells and fed a high cholesterol diet. Jnk1(-/-)?Ldlr(-/-) mice have larger atherosclerotic lesions with more macrophages and fewer apoptotic cells than mice transplanted with wild-type or Jnk2(-/-) cells. Moreover, genetic ablation of JNK to a single allele (Jnk1(+/-)/Jnk2(-/-) or Jnk1(-/-)/Jnk2(+/-)) in marrow of Ldlr(-/-) recipients further increased atherosclerosis compared with Jnk1(-/-)?Ldlr(-/-) and wild-type?Ldlr(-/-) mice. In mouse macrophages, anisomycin-mediated JNK signaling antagonized Akt activity, and loss of Jnk1 gene obliterated this effect. Similarly, pharmacological inhibition of JNK1, but not JNK2, markedly reduced the antagonizing effect of JNK on Akt activity. Prolonged JNK signaling in the setting of endoplasmic reticulum stress gradually extinguished Akt and Bad activity in wild-type cells with markedly less effects in Jnk1(-/-) macrophages, which were also more resistant to apoptosis. Consequently, anisomycin increased and JNK1 inhibitors suppressed endoplasmic reticulum stress-mediated apoptosis in macrophages. We also found that genetic and pharmacological inhibition of phosphatase and tensin homolog abolished the JNK-mediated effects on Akt activity, indicating that phosphatase and tensin homolog mediates crosstalk between these pathways.Loss of Jnk1, but not Jnk2, in macrophages protects them from apoptosis, increasing cell survival, and this accelerates early atherosclerosis.
Project description:Forkhead box O1 (FoxO1) is a transcription factor that mediates the inhibitory effect of insulin on target genes in hepatic metabolism. Hepatic FoxO1 activity is up-regulated to promote glucose production during fasting and is suppressed to limit postprandial glucose excursion after meals. Increased FoxO1 activity augments the expression of insulin receptor (IR) and IR substrate (IRS)2, which in turn inhibits FoxO1 activity in response to reduced insulin action. To address the underlying physiology of such a feedback loop for regulating FoxO1 activity, we delivered FoxO1-ADA by adenovirus-mediated gene transfer into livers of adult mice. FoxO1-ADA is a constitutively active allele that is refractory to insulin inhibition, allowing us to determine the metabolic effect of a dislodged FoxO1 feedback loop in mice. We show that hepatic FoxO1-ADA production resulted in significant induction of IR and IRS2 expression. Mice with increased FoxO1-ADA production exhibited near glycogen depletion. Unexpectedly, hepatic FoxO1-ADA production elicited a profound unfolded protein response, culminating in the induction of hepatic glucose-regulated protein 78 (GRP78) expression. These findings were recapitulated in primary human and mouse hepatocytes. FoxO1 targeted GRP78 gene for trans-activation via selective binding to an insulin responsive element in the GRP78 promoter. This effect was counteracted by insulin. Our studies underscore the importance of an IR and IRS2-dependent feedback loop to keep FoxO1 activity in check for maintaining hepatic glycogen homeostasis and promoting adaptive unfolded protein response in response to altered metabolism and insulin action. Excessive FoxO1 activity, resulting from a dislodged FoxO1 feedback loop in insulin resistant liver, is attributable to hepatic endoplasmic reticulum stress and metabolic abnormalities in diabetes.
Project description:Chronic inflammation in adipose tissue contributes to obesity-related insulin resistance. The 3-phosphoinositide-dependent protein kinase 1 (Pdk1)/forkhead transcription factor (Foxo1) pathway is important in regulating glucose and energy homeostasis, but little is known about this pathway in adipose tissue macrophages (ATMs). To investigate this, we generated transgenic mice that carried macrophage/granulocyte-specific mutations, including a Pdk1 knockout (LysMPdk1(-/-)), a Pdk1 knockout with transactivation-defective Foxo1 (?256LysMPdk1(-/-)), a constitutively active nuclear (CN) Foxo1 (CNFoxo1(LysM)), or a transactivation-defective Foxo1 (?256Foxo1(LysM)). We analyzed glucose metabolism and gene expression in ATM populations isolated with fluorescence-activated cell sorting. The LysMPdk1(-/-) mice exhibited elevated M1 macrophages in adipose tissue and insulin resistance. Overexpression of transactivation-defective Foxo1 rescued these phenotypes. CNFoxo1(LysM) promoted transcription of the C-C motif chemokine receptor 2 (Ccr2) in ATMs and increased M1 macrophages in adipose tissue. On a high-fat diet, CNFoxo1(LysM) mice exhibited insulin resistance. Pdk1 deletion or Foxo1 activation in bone marrow-derived macrophages abolished insulin and interleukin-4 induction of genes involved in alternative macrophage activation. Thus, Pdk1 regulated macrophage infiltration by inhibiting Foxo1-induced Ccr2 expression. This shows that the macrophage Pdk1/Foxo1 pathway is important in regulating insulin sensitivity in vivo.
Project description:Obesity-mediated inflammation is a major cause of insulin resistance, and macrophages play an important role in this process. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum chaperone that modulates unfolded protein response (UPR), and mice with GRP78 heterozygosity were resistant to diet-induced obesity. Here, we show that mice with macrophage-selective ablation of GRP78 (Lyz- GRP78-/-) are protected from skeletal muscle insulin resistance without changes in obesity compared with wild-type mice after 9 wk of high-fat diet. GRP78-deficient macrophages demonstrated adapted UPR with up-regulation of activating transcription factor (ATF)-4 and M2-polarization markers. Diet-induced adipose tissue inflammation was reduced, and bone marrow-derived macrophages from Lyz- GRP78-/- mice demonstrated a selective increase in IL-6 expression. Serum IL-13 levels were elevated by >4-fold in Lyz- GRP78-/- mice, and IL-6 stimulated the myocyte expression of IL-13 and IL-13 receptor. Lastly, recombinant IL-13 acutely increased glucose metabolism in Lyz- GRP78-/- mice. Taken together, our data indicate that GRP78 deficiency activates UPR by increasing ATF-4, and promotes M2-polarization of macrophages with a selective increase in IL-6 secretion. Macrophage-derived IL-6 stimulates the myocyte expression of IL-13 and regulates muscle glucose metabolism in a paracrine manner. Thus, our findings identify a novel crosstalk between macrophages and skeletal muscle in the modulation of obesity-mediated insulin resistance.-Kim, J. H., Lee, E., Friedline, R. H., Suk, S., Jung, D. Y., Dagdeviren, S., Hu, X., Inashima, K., Noh, H. L., Kwon, J. Y., Nambu, A., Huh, J. R., Han, M. S., Davis, R. J., Lee, A. S., Lee, K. W., Kim, J. K. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.
Project description:Oxidized HDL (ox-HDL), unlike native HDL that exerts antiatherogenic effects, plays a proatherogenic role. However, the underlying mechanisms are not completely understood. This study was designed to explore the inductive effect of ox-HDL on endoplasmic reticulum (ER) stress-CCAAT-enhancer-binding protein homologous protein (CHOP)-mediated macrophage apoptosis and its upstream mechanisms. Our results showed that ox-HDL could be ingested by macrophages, causing intracellular lipid accumulation. As with tunicamycin (an ER stress inducer), ox-HDL induced macrophage apoptosis with concomitant activation of ER stress pathway, including nuclear translocation of activating transcription factor 6, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2?, and upregulation of glucose-regulated protein 78 and CHOP, which were inhibited by 4-phenylbutyric acid (PBA, an ER stress inhibitor) and CHOP gene silencing. Additionally, diphenyleneiodonium (DPI, an oxidative stress inhibitor), probucol (a reactive oxygen species scavenger), and toll-like receptor 4 (TLR4) silencing reduced ox-HDL-induced macrophage apoptosis, oxidative stress, and CHOP upregulation. Moreover, HDL isolated from patients with metabolic syndrome induced macrophage apoptosis, oxidative stress, and CHOP upregulation, which were blocked by PBA and DPI. These data indicate that ox-HDL may activate ER stress-CHOP-induced apoptotic pathway in macrophages via enhanced oxidative stress and that this pathway may be mediated by TLR4.
Project description:This study was designed to explore the inductive effect of glycated high-density lipoprotein (gly-HDL) on endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP)-mediated macrophage apoptosis and its relationship with autophagy. Our results showed that gly-HDL caused macrophage apoptosis with concomitant activation of ER stress pathway, including nuclear translocation of activating transcription factor 6, phosphorylation of protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2?, and CHOP up-regulation, which were inhibited by 4-phenylbutyric acid (PBA, an ER stress inhibitor) and the gene silencing of PERK and CHOP. Similar data were obtained from macrophages treated by HDL isolated from diabetic patients. Gly-HDL induced macrophage autophagy as assessed by up-regulation of beclin-1, autophagy-related gene 5 and microtubule-associated protein one light chain 3-II, which were depressed by PBA and PERK siRNA. Gly-HDL-induced apoptosis, PERK phosphorylation and CHOP up-regulation were suppressed by rapamycin (an autophagy inducer), whereas aggravated by 3-methyladenine (an autophagy inhibitor) and beclin-1 siRNA. Administration of diabetic apoE-/- mice with rapamycin attenuated MOMA-2 and CHOP up-regulation and apoptosis in atherosclerotic lesions. These data indicate that gly-HDL may induce macrophage apoptosis through activating ER stress-CHOP pathway and ER stress mediates gly-HDL-induced autophagy, which in turn protects macrophages against apoptosis by alleviating CHOP pathway.